Inhibitors of RET

ABSTRACT

Described herein are compounds that inhibit wild-type RET and its resistant mutants, pharmaceutical compositions including such compounds, and methods of using such compounds and compositions.

CLAIM OF PRIORITY

This application claims priority from U.S. Ser. No. 62/309,748, filedMar. 17, 2016, which is incorporated herein in its entirety.

This invention relates to inhibitors of RET that are active againstwild-type RET and its resistant mutants.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2017, isnamed B2094-7022WO_SL.txt and is 32,316 bytes in size.

BACKGROUND

RET (rearranged during transfection) is a receptor tyrosine kinase thatactivates multiple downstream pathways involved in cell proliferationand survival. RET fusions are implicated in several cancers includingpapillary thyroid carcinoma and non-small cell lung cancer. A genomicsanalysis on the landscape of kinase fusions identified RET fusions inbreast and colon cancer patient samples, providing therapeutic rationalefor the use of RET inhibitors in multiple patient subpopulations.

The identification of RET fusions as drivers in some cancers promptedthe use of approved multi-kinase inhibitors with RET inhibitory activityto treat patients whose tumors express a RET fusion protein. However,these drugs cannot always be dosed at the levels required tosufficiently inhibit RET due to toxicities that result from inhibitionof targets other than RET. Further, one of the greatest challenges intreating cancer is the ability of tumor cells to become resistant totherapy. Kinase reactivation via mutation is a common mechanism ofresistance. When resistance occurs, the patient's treatment options areoften very limited, and the cancer progresses, unchecked, in mostinstances. There is thus a need for compounds that inhibit RET, as wellas its resistant mutants.

SUMMARY

The present invention provides inhibitors of RET and RET mutants, e.g.,RET resistant mutants (as defined herein), for example, inhibitors ofstructural Formula (I) and pharmaceutically acceptable salts andcompositions thereof. The present invention further provides methods ofusing the compounds of the invention, and pharmaceutically acceptablesalts and compositions thereof, to inhibit the activity of RET or RETmutants in a cell or patient. The present invention still furtherprovides methods for using the compounds of the invention, andpharmaceutically acceptable salts and compositions thereof, to treat asubject suffering from a condition mediated by aberrant RET activity,e.g., cancer.

In one aspect, the invention features a compound of structural Formula(I) or a pharmaceutically acceptable salt thereof:

wherein each of ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷,R^(8a), R^(8b), R⁹, m and n is defined as described herein.

In another aspect, the present invention provides pharmaceuticalcompositions comprising a compound of structural Formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In another aspect, the present invention provides a method forinhibiting RET activity in a cell or in a patient. In some embodiments,said method comprises the step of contacting the cell or administeringto the patient a compound of structural Formula (I) or apharmaceutically acceptable salt or composition thereof. In someembodiments, the cell or patient has, or has been identified as having,a RET alteration, e.g., a RET mutation, e.g., a fusion or pointmutation. In some embodiments, the patient comprises a RET-altered cell,cancer, gene, or gene product.

In another aspect, the present invention provides a method for treatinga subject suffering from a condition mediated by aberrant RET activity.In some embodiments, said method comprises administering to the subjecta therapeutically effective amount of a compound of structural Formula(I) or a pharmaceutically acceptable salt or composition thereof. Insome embodiments, the subject has or has been identified as having(e.g., wherein a cancer cell in the subject has or has been identifiedas having) a RET alteration, e.g., a RET mutation, e.g., a fusion orpoint mutation. In some embodiments, a cell, cancer, gene, or geneproduct from the subject is or has been identified as being RET-altered.In some embodiments, the subject has or has been identified as having(e.g., a cancer cell in the subject has or has been identified ashaving) a RET alteration, e.g., a RET mutation, e.g., a fusion or pointmutation.

In some embodiments, the condition mediated by aberrant RET activity isa condition mediated by any RET activity that is not normal e.g., anyactivity due to a RET-altered gene or gene product, which affects theamount or activity of the gene or gene product as compared to the normalor wild-type gene. In embodiments, the condition mediated by aberrantRET activity is a familial or sporadic cancer, e.g., a solid tumor suchas thyroid, lung, breast, or pancreatic. In embodiments, the conditionmediated by aberrant RET activity is irritable bowel syndrome (IBS). Inembodiments, the aberrant RET activity promotes the condition, such thatinhibition of RET ameliorates at least one symptom of the condition. Inembodiments, the aberrant RET activity comprises increased RET activityor expression level, gain of function mutation, and/or constitutiveactivation of RET. In embodiments, the aberrant RET activity correspondsto aberrant amounts of RET, e.g., aberrant nucleic acid or proteinamounts.

In another aspect, the present invention provides a method for treatinga subject who has developed resistance to a cancer treatment. In someembodiments, said method comprises administering to the subject atherapeutically effective amount of a compound of structural Formula (I)or a pharmaceutically acceptable salt or composition thereof. In someembodiments, the subject has developed resistance to a wild-type RETinhibitor. In embodiments, the cancer treatment to which the subject isresistant is a wild-type RET inhibitor that is active against thewild-type RET, but less active, e.g., much less active, against one ormore mutated forms of RET. In some embodiments, the wild-type RETinhibitor is selected from ponatinib, carbozanitib, and vandetanib.

In another aspect, the present invention provides a use of a compound orpharmaceutical composition described herein, e.g., a compound ofstructural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein(e.g., a compound in Table 1) or a pharmaceutically acceptable salt orcomposition thereof, in the manufacture of a medicament for inhibitingRET activity in a cell or in a subject. In embodiments, the cell orsubject has, or has been identified as having, a RET alteration, e.g., aRET mutation, e.g., a fusion or point mutation. In embodiments, a cell,cancer, gene, or gene product from the subject is or has been identifiedas being RET-altered.

In another aspect, the present invention provides a use of a compound orpharmaceutical composition described herein, e.g., a compound ofstructural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein(e.g., a compound in Table 1) or a pharmaceutically acceptable salt orcomposition thereof, in the manufacture of a medicament for treating asubject suffering from a condition mediated by aberrant RET activity. Inembodiments, the subject has, or has been identified as having (e.g.,wherein a cancer cell in the subject has, or has been identified ashaving) a RET alteration, e.g., a RET mutation, e.g., a fusion or pointmutation. In embodiments, a cell, cancer, gene, or gene product from thesubject is or has been identified as being RET-altered.

In another aspect, the present invention provides a use of a compound orpharmaceutical composition described herein, e.g., a compound ofstructural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein(e.g., a compound in Table 1) or a pharmaceutically acceptable salt orcomposition thereof, in the manufacture of a medicament for treating asubject who has developed resistance to a cancer treatment.

In another aspect, the present invention provides a method of preventingdevelopment of one or more RET-altered cell, cancer, gene, or geneproduct, in a cell or in a patient, comprising the step of contactingthe cell or administering to the patient a compound according tostructural Formula (I).

In another aspect, the present invention provides a method of treating asubject suffering from cancer comprising the steps of:

-   -   a. receiving information related to a RET sequence, e.g.,        information related to a RET-altered gene or gene product, e.g.,        having a RET fusion or point mutation; and    -   b. administering to the subject a compound described herein        (e.g., a compound having a lower IC₅₀ for RET than for KDR, a        compound having a similar IC₅₀ for wild-type RET compared to        mutant RET, and/or a compound of structural Formula I, (Ia),        (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in        Table 1)), or a pharmaceutical composition comprising the        compound, if the information indicates a RET-altered cell,        cancer, gene, or gene product.

In embodiments, the subject is administered a cancer treatment otherthan a compound of structural Formula (I) or (II) if the informationindicates that the subject has a wild-type RET sequence. In embodiments,the cancer treatment is a wild-type RET inhibitor.

In another aspect, the present invention provides a method of treatingcancer in a subject, said method comprising:

a. obtaining a biological sample (e.g., a tumor biopsy) from a humansubject;

b. detecting whether a RET-altered cell, cancer, gene, or gene product,e.g., having a fusion or point mutation, is present in the biologicalsample;

c. identifying the subject as responsive to a compound described herein(e.g., a compound having a lower IC₅₀ for RET than for KDR, a compoundhaving a similar IC₅₀ for wild-type RET compared to mutant RET, and/or acompound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb)described herein (e.g., a compound in Table 1)) when the presence of theRET-altered cell, cancer, gene, or gene product in the biological sampleis detected; and

d. administering an effective amount of the compound to the subject.

In another aspect, the present invention provides a method of treatingcancer in a subject, said method comprising:

-   -   a. determining if, having determined if, or receiving        information that the subject has a RET-altered cell, cancer,        gene, or gene product, e.g., having a fusion or point mutation;    -   b. identifying the subject as responsive to a compound described        herein (e.g., a compound having a lower IC₅₀ for RET than for        KDR, a compound having a similar IC₅₀ for wild-type RET compared        to mutant RET, and/or a compound of structural Formula I, (Ia),        (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in        Table 1)) when the subject has a RET-altered cell, cancer, gene,        or gene product; and    -   c. administering an effective amount of the compound to the        subject.

In another aspect, the present invention provides a method of diagnosingcancer in a subject, said method comprising:

a. obtaining a biological sample (e.g., a tumor biopsy) from a humansubject;

b. detecting whether a RET-altered cell, cancer, gene, or gene product,e.g., having a fusion or point mutation, is present in the biologicalsample;

c. diagnosing the subject with cancer when the presence of theRET-altered cell, cancer, gene, or gene product in the biological sampleis detected.

In another aspect, the present invention provides a method of predictingthe efficacy of a compound described herein (a compound having a lowerIC₅₀ for RET than for KDR, a compound having a similar IC₅₀ forwild-type RET compared to mutant RET, and/or a compound of structuralFormula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., acompound in Table 1)) in a treatment of cancer in a subject comprisingthe step of:

determining if, having determined if, or receiving information that thesubject has a RET-altered cell, cancer, gene, or gene product, e.g.,having a mutation, e.g., a fusion or point mutation, e.g., by a methodselected from hybridization-based methods, amplification-based methods,microarray analysis, flow cytometry analysis, DNA sequencing,next-generation sequencing (NGS), primer extension, PCR, in situhybridization, dot blot, and Southern blot;

wherein said determining if, having determined if, or receivinginformation is predictive of efficacy of the compound in the treatment.

In some embodiments of any of the methods and uses herein, the methodfurther comprises administering to the subject a compound describedherein, e.g., (e.g., a compound having a lower IC₅₀ for RET than forKDR, a compound having a similar IC₅₀ for wild-type RET compared tomutant RET, and/or a compound of structural Formula I, (Ia), (Ib), II,(IIa), or (IIb) described herein (e.g., a compound in Table 1)), e.g.,responsive to a determination or diagnosis made by a method describedherein.

In some embodiments of any of the methods and uses herein, theRET-altered cell, cancer, gene, or gene product comprises a fusionmutation, e.g., CCDC6-RET or KIF5B-RET or a fusion of Table 3. Inembodiments, the RET-altered cell, cancer, gene, or gene productcomprises a point mutation of amino acid position 634, 918, or 804 ofRET, or a point mutation at a position listed in Table 4, e.g., a pointmutation specified in Table 4. In some embodiments, the RET-alteredcell, cancer, gene, or gene product comprises a point mutation at aminoacid 634, 918, or 804, 806, 810, 865, 870, 891, e.g., is selected fromRET C634W, M918T, V804L, V804E, V804M, V804L, V806C, Y806C, Y806S,Y806N, Y806H, G810R, G810S, L865V, L870F, and S891A. In someembodiments, the RET alteration is located at least partially within, oris located wholly within one or more of: the N-terminal extracellulardomain (e.g., within one or more cadherin-like repeats and/or thecysteine-rich region), the transmembrane domain, or the tyrosine kinasedomain (e.g., within one or more of the ATP binding site or protonacceptor site). In some embodiments, the RET fusion further comprises apoint mutation, e.g., KIF5B-RET (V804L) or KIF5B-RET (V804M).

In some embodiments of any of the methods and uses herein, the subjectsuffers from a cancer selected from colorectal cancer, lung cancer(e.g., a lung adenocarcinoma, e.g., NSCLC), thyroid cancer (e.g.,medullary thyroid cancer), or leukemia. In embodiments, the subjectsuffers from a cancer listed herein, e.g., in Table 3.

In some embodiments, the cancer is wild-type RET.

In some embodiments, the cancer is lung cancer, e.g., a lungadenocarcinoma, and the RET-altered cell, cancer, gene, or gene productcomprises a CCDC6-RET fusion. In some embodiments, the cancer is lungadenocarcinoma and the RET-altered cell, cancer, gene, or gene productcomprises a KIF5B-RET fusion. In some embodiments, the cancer is lungadenocarcinoma and the RET-altered cell, cancer, gene, or gene productcomprises KIF5B-RET (V804L). In some embodiments, the lungadenocarcinoma is NSCLC.

In some embodiments, the cancer is thyroid cancer, e.g., medullarythyroid cancer, and the RET-altered cell, cancer, gene, or gene productcomprises a C634W mutation.

In some embodiments, the cancer is leukemia and the RET-altered cell,cancer, gene, or gene product comprises a KIF5B-RET fusion.

In some embodiments, the cancer is thyroid cancer and the RET-alteredcell, cancer, gene, or gene product comprises a M918T mutation. In someembodiments, the cancer is medullary thyroid cancer and the RET-alteredcell, cancer, gene, or gene product comprises a M918T mutation.

In some embodiments, the cancer is leukemia and the RET-altered cell,cancer, gene, or gene product comprises KIF5B-RET (V804L) or KIF5B-RET(V804M) fusion.

In some embodiments, the cancer is thyroid cancer and the RET-alteredcell, cancer, gene, or gene product comprises a CCDC6-RET fusion. Insome embodiments, the cancer is colorectal cancer and the RET-alteredcell, cancer, gene, or gene product comprises CCDC6-RET fusion. In someembodiments, the cancer is colorectal cancer and the RET-altered cell,cancer, gene, or gene product comprises CCDC6-RET (V804M) fusion.

In some embodiments of any of the methods and uses herein, the compoundor pharmaceutical composition has a lower IC₅₀ for RET than for KDR,e.g., has a KDR/RET IC₅₀ ratio of at least 3×, 4×, 5×, 10×, 20×, 50×, or100×, and optionally up to 50× or 100×. In embodiments, the compound orpharmaceutical composition has a KDR/RET IC₅₀ ratio of between 3×-4×,4×-5×, 5×-10×, 10×-20×, 20×-50×, or 50×-100×. In some embodiments, thecompound or pharmaceutical composition has a similar IC₅₀ for wild-typeRET compared to mutant RET (e.g., for V804L RET or V804E RET), e.g., hasa wild-type/mutant IC₅₀ ratio of no more than 3×, 2×, 1.5×, 1×, or 0.5×,e.g., of between 3× and 0.5×.

In some embodiments of any of the methods and uses herein, the subjectdoes not develop a RET-altered cell, cancer, gene, or gene product forat least 1, 2, 3, 6, 9, 12, 24, or 36 months after initiation ofadministration of the compound. In some embodiments, the compound isadministered as a first line therapy. In some embodiments, the compoundis administered to a treatment-naïve subject. In some embodiments, thecompound is not administered in combination with another kinaseinhibitor. In some embodiments, the compound is not administered incombination with another RET inhibitor. For example, the compound can beadministered as a monotherapy or in combination with one or other agentswhich are not kinase inhibitors, e.g., not RET inhibitors.

In some embodiments of any of the methods and uses herein, the compoundis administered at an amount sufficient to reach at least 70%, 80%, 90%,or 95% inhibition of RET in vivo.

EMBODIMENTS OF THE INVENTION Definitions

As used herein, the terms a “patient,” “subject,” “individual,” and“host” refer to either a human or a non-human animal suffering from orsuspected of suffering from a disease or disorder associated withaberrant RET expression (i.e., increased RET activity caused bysignaling through RET) or biological activity.

“Treat” and “treating” such a disease or disorder refers to amelioratingat least one symptom of the disease or disorder. These terms, when usedin connection with a condition such as a cancer, refer to one or moreof: impeding growth of the cancer, causing the cancer to shrink byweight or volume, extending the expected survival time of the patient,inhibiting tumor growth, reducing tumor mass, reducing size or number ofmetastatic lesions, inhibiting the development of new metastaticlesions, prolonging survival, prolonging progression-free survival,prolonging time to progression, and/or enhancing quality of life.

The term “therapeutic effect” refers to a beneficial local or systemiceffect in animals, particularly mammals, and more particularly humans,caused by administration of a compound or composition of the invention.The phrase “therapeutically-effective amount” means that amount of acompound or composition of the invention that is effective to treat adisease or condition caused by over expression of RET or aberrant RETbiological activity at a reasonable benefit/risk ratio. Thetherapeutically effective amount of such substance will vary dependingupon the subject and disease or condition being treated, the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofskill in the art.

As used herein, “developing resistance” means that when a drug is firstadministered to the patient, the patient's symptoms improve, whethermeasured by decrease in tumor volume, a decrease in the number of newlesions, or some other means that a physician uses to judge diseaseprogression; however, those symptoms stop improving, or even worsen atsome point. At that time, the patient is said to have developedresistance to the drug.

“Alteration” as used herein, of a gene or gene product (e.g., the RETgene or gene product) refers to the presence of a mutation or mutationswithin the gene or gene product, e.g., a mutation, which of the gene orgene product, as compared to the normal or wild-type gene. Thealteration can be in amount, structure, and/or activity in a cancertissue or cancer cell, as compared to its amount, structure, and/oractivity, in a normal or healthy tissue or cell (e.g., a control), andis associated with a disease or condition, such as cancer. For example,an alteration which is associated with cancer, or predictive ofresponsiveness to an anti-cancer therapeutic, can have an alterednucleotide sequence (e.g., a mutation), amino acid sequence, chromosomaltranslocation, intra-chromosomal inversion, copy number, expressionlevel, protein level, protein activity, or methylation status, in acancer tissue or cancer cell, as compared to a normal, healthy tissue orcell. Exemplary mutations include, but are not limited to, pointmutations (e.g., silent, missense, or nonsense), deletions, insertions,inversions, linking mutations, duplications, translocations, inter- andintra-chromosomal rearrangements. Mutations can be present in the codingor non-coding region of the gene, e.g., a 3′ UTR or 5′ UTR.

A subject having “altered RET” refers to a subject comprising a RETalteration, e.g., in one or more of their cancer cells.

A “RET-altered” cell, cancer, gene, or gene product refers to a cell,cancer, gene, or gene product comprising a RET alteration as describedherein.

“Aliphatic group” means a straight-chain, branched-chain, or cyclichydrocarbon group and includes saturated and unsaturated groups, such asan alkyl group, an alkenyl group, and an alkynyl group.

“Alkylene” refers to a divalent radical of an alkyl group, e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—.

“Alkenyl” means an aliphatic group containing at least one double bond.

“Alkoxyl” or “alkoxy” means an alkyl group having an oxygen radicalattached thereto. Representative alkoxyl groups include methoxy, ethoxy,propyloxy, tert-butoxy and the like. The term “haloalkoxy” refers to analkoxy in which one or more hydrogen atoms are replaced by halo, andincludes alkoxy moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkoxy).

“Alkyl” refers to a monovalent radical of a saturated straight orbranched hydrocarbon, such as a straight or branched group of 1-12,1-10, or 1-6 carbon atoms, referred to herein as C₁-C₁₂ alkyl, C₁-C₁₀alkyl, and C₁-C₆ alkyl, respectively. Exemplary alkyl groups include,but are not limited to, methyl, ethyl, propyl, isopropyl,2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl,3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl,isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl,etc.

“Alkenylene” refers to an alkenyl group having two connecting points.For example, “ethenylene” represents the group —CH═CH—. Alkenylenegroups can also be in an unsubstituted form or substituted form with oneor more substituents.

“Alkynyl” refers to a straight or branched hydrocarbon chain containing2-12 carbon atoms and characterized in having one or more triple bonds.Examples of alkynyl groups include, but are not limited to, ethynyl,propargyl, and 3-hexynyl. One of the triple bond carbons may optionallybe the point of attachment of the alkynyl substituent.

“Alkynylene” refers to an alkynyl having two connecting points. Forexample, “ethynylene” represents the group —C≡C—. Alkynylene groups canalso be in an unsubstituted form or substituted form with one or moresubstituents.

“Aromatic ring system” is art-recognized and refers to a monocyclic,bicyclic or polycyclic hydrocarbon ring system, wherein at least onering is aromatic.

“Aryl” refers to a monovalent radical of an aromatic ring system.Representative aryl groups include fully aromatic ring systems, such asphenyl, naphthyl, and anthracenyl, and ring systems where an aromaticcarbon ring is fused to one or more non-aromatic carbon rings, such asindanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and thelike.

“Arylene” refers to a divalent aryl, wherein “aryl” is as definedherein.

“Arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkylhydrogen atom is replaced by an aryl group. Aralkyl includes groups inwhich more than one hydrogen atom has been replaced by an aryl group.Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl,3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

“Aryloxy” refers to —O-(aryl), wherein the aryl moiety is as definedherein.

“Halo” refers to a radical of any halogen, e.g., —F, —Cl, —Br, or —I.

“Heteroalkyl” refers to an optionally substituted alkyl, which has oneor more skeletal chain atoms selected from an atom other than carbon,e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Anumerical range may be given, e.g. C₁-C₆ heteroalkyl which refers to thenumber of carbons in the chain, which in this example includes 1 to 6carbon atoms. For example, a —CH₂OCH₂CH₃ radical is referred to as a“C₃” heteroalkyl. Connection to the rest of the molecule may be througheither a heteroatom or a carbon in the heteroalkyl chain.

“Heteroalkylene” refers to a divalent optionally substituted alkyl,which has one or more skeletal chain atoms selected from an atom otherthan carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinationsthereof.

“Carbocyclic ring system” refers to a monocyclic, bicyclic or polycyclichydrocarbon ring system, wherein each ring is either completelysaturated or contains one or more units of unsaturation, but where noring is aromatic.

“Carbocyclyl” refers to a monovalent radical of a carbocyclic ringsystem. Representative carbocyclyl groups include cycloalkyl groups(e.g., cyclobutyl, cyclopentyl, cyclohexyl and the like), andcycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl,cyclopentadienyl, and the like).

“Cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclicnon-aromatic hydrocarbon groups having 3 to 12 carbons. Anysubstitutable ring atom can be substituted (e.g., by one or moresubstituents). The cycloalkyl groups can contain fused or spiro rings.Fused rings are rings that share a common carbon atom. Examples ofcycloalkyl moieties include, but are not limited to, cyclopropyl,cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.

“Cycloalkylalkyl” refers to a -(cycloalkyl)-alkyl radical wherecycloalkyl and alkyl are as disclosed herein. The “cycloalkylalkyl” isbonded to the parent molecular structure through the cycloalkyl group.

“Heteroaromatic ring system” is art-recognized and refers to monocyclic,bicyclic or polycyclic ring system wherein at least one ring is botharomatic and comprises at least one heteroatom (e.g., N, O or S); andwherein no other rings are heterocyclyl (as defined below). In certaininstances, a ring which is aromatic and comprises a heteroatom contains1, 2, 3, or 4 ring heteroatoms in such ring.

“Heteroaryl” refers to a monovalent radical of a heteroaromatic ringsystem. Representative heteroaryl groups include ring systems where (i)each ring comprises a heteroatom and is aromatic, e.g., imidazolyl,oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl,pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl; (ii) each ring is aromatic orcarbocyclyl, at least one aromatic ring comprises a heteroatom and atleast one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, pyrido[2,3-b]-1,4-oxazin-3-(4H)-one,5,6,7,8-tetrahydroquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl; and(iii) each ring is aromatic or carbocyclyl, and at least one aromaticring shares a bridgehead heteroatom with another aromatic ring, e.g.,4H-quinolizinyl.

“Heteroarylene” refers to a divalent heteroaryl, wherein “heteroaryl” isas defined herein.

“Heterocyclic ring system” refers to monocyclic, bicyclic and polycyclicring systems where at least one ring is saturated or partiallyunsaturated (but not aromatic) and that ring comprises at least oneheteroatom. A heterocyclic ring system can be attached to its pendantgroup at any heteroatom or carbon atom that results in a stablestructure and any of the ring atoms can be optionally substituted.

“Heterocyclyl” refers to a monovalent radical of a heterocyclic ringsystem. Representative heterocyclyls include ring systems in which (i)every ring is non-aromatic and at least one ring comprises a heteroatom,e.g., tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl,pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl;(ii) at least one ring is non-aromatic and comprises a heteroatom and atleast one other ring is an aromatic carbon ring, e.g.,1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii)at least one ring is non-aromatic and comprises a heteroatom and atleast one other ring is aromatic and comprises a heteroatom, e.g.,3,4-dihydro-1H-pyrano[4,3-c]pyridine, and1,2,3,4-tetrahydro-2,6-naphthyridine.

“Heterocyclylalkyl” refers to an alkyl group substituted with aheterocyclyl group.

“Cyano” refers to a —CN radical.

“Nitro” refers to —NO₂.

“Hydroxy” or “hydroxyl” refers to —OH.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.Thus, when a disclosed compound is named or depicted by a structurewithout specifying the stereochemistry and has one or more chiralcenters, it is understood to represent all possible stereoisomers of thecompound, as well as enantiomeric mixtures thereof. When a disclosedcompound is named or depicted by a structure specifying stereochemistryat each chiral center, it is understood to represent only the compoundhaving the designated stereochemistry at such chiral centers. However,when a disclosed compound specifies stereochemistry at some, but not allchiral centers, it is understood to represent all possible stereoisomersat the non-specified chiral centers of the compound, as well asenantiomeric mixtures thereof.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The “enantiomeric excess” or “% enantiomeric excess” of a compositioncan be calculated using the equation shown below. In the example shownbelow a composition contains 90% of one enantiomer, e.g., the Senantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.ee=(90−10)/100=80%.

Thus, a composition containing 90% of one enantiomer and 10% of theother enantiomer is said to have an enantiomeric excess of 80%.

The compounds or compositions described herein may contain anenantiomeric excess of at least 50%, 75%, 90%, 95%, or 99% of one formof the compound, e.g., the S-enantiomer. In other words such compoundsor compositions contain an enantiomeric excess of the S enantiomer overthe R enantiomer.

The compounds described herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example deuterium (²H), tritium (³H),carbon-13 (¹³C), or carbon-14 (¹⁴C). All isotopic variations of thecompounds disclosed herein, whether radioactive or not, are intended tobe encompassed within the scope of the present invention. In addition,all tautomeric forms of the compounds described herein are intended tobe within the scope of the invention.

The compound can be useful as the free base or as a salt. Representativesalts include the hydrobromide, hydrochloride, sulfate, bisulfate,phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts and the like. (See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19.)

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at eachposition. Combinations of substituents envisioned under this inventionare preferably those that result in the formation of stable orchemically feasible compounds. The term “stable”, as used herein, refersto compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable substituents for an optionally substituted alkyl, alkylene,heteroalkyl, heteroalkylene, carbocyclyl, heterocyclyl, aryl group andheteroaryl group include halogen, ═O, —CN, —OR^(c), —NR^(d)R^(e),—S(O)_(k)R^(c), —NR^(c)S(O)₂R^(c), —S(O)₂NR^(d)R^(e), —C(═O)OR^(c),—OC(═O)OR^(c), —OC(═O)R^(c), —OC(═S)OR^(c), —C(═S)OR^(c), —O(C═S)R^(c),—C(═O)NR^(d)R^(e), —NR^(c)C(═O)R^(c), —C(═S)NR^(d)R^(e),—NR^(c)C(═S)R^(c), —NR^(c)(C═O)OR^(c), —O(C═O)NR^(d)R^(e),—NR^(c)(C═S)OR^(c), —O(C═S)NR^(d)R^(e), —NR^(c)(C═O)NR^(d)R^(e),—NR^(c)(C═S)NR^(d)R^(e), —C(═S)R^(c), —C(═O)R^(c), C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl, carbocyclyl, (C₁-C₆-alkylene)-carbocyclyl,(C₁-C₆-heteroalkylene)-carbocyclyl, heterocyclyl,(C₁-C₆-alkylene)-heterocyclyl, (C₁-C₆-heteroalkylene)-heterocyclyl,aryl, (C₁-C₆-alkylene)-aryl, (C₁-C₆-heteroalkylene)-aryl, heteroaryl,(C₁-C₆-alkylene)-heteroaryl, or (C₁-C₆-heteroalkylene)-heteroaryl,wherein each of said alkyl, alkylene, heteroalkyl, heteroalkylene,carbocyclyl, heterocyclyl, aryl and heteroaryl are optionallysubstituted with one or more of halogen,

OR^(c), —NO₂, —CN, —NR^(c)C(═O)R^(c), —NR^(d)R^(e), —S(O)_(k)R^(c),—C(═O)OR^(c), —C(═O)NR^(d)R^(e), —C(═O)R^(c), C₁-C₆ alkyl, C₁-C₆haloalkyl, or C₁-C₆ heteroalkyl, and wherein R^(c) is hydrogen, hydroxy,C₁-C₆ alkyl, C₁-C₆ heteroalkyl, carbocyclyl,(C₁-C₆-alkylene)-carbocyclyl, (C₁-C₆-heteroalkylene)-carbocyclyl,heterocyclyl, (C₁-C₆-alkylene)-heterocyclyl,(C₁-C₆-heteroalkylene)-heterocyclyl, aryl, (C₁-C₆-alkylene)-aryl,(C₁-C₆-heteroalkylene)-aryl, heteroaryl, (C₁-C₆-alkylene)-heteroaryl, or(C₁-C₆-heteroalkylene)-heteroaryl, each of which is optionallysubstituted with one or more of halogen, hydroxy, C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ heteroalkyl, carbocyclyl, heterocyclyl, aryl, orheteroaryl; R^(d) and R^(e) are each independently selected fromhydrogen, C₁-C₆ alkyl, or C₁-C₆ heteroalkyl; and k is 0, 1 or 2. Theinvention is not intended to be limited in any manner by the aboveexemplary listing of substituents.Compounds

In one aspect, the present invention features a compound having thestructural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

ring A is an aryl or heteroaryl ring;

each of X¹ and X² is independently selected from N and C(R⁶);

each R¹ and each R⁷ is independently selected from selected from C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, halo, C₁-C₆heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl,heterocyclyl, heterocyclylalkyl, nitro, cyano, —C(O)R, —OC(O)R, —C(O)OR,—(C₁-C₆ alkylene)-C(O)R, —SR, —S(O)₂R, —S(O)₂—N(R)(R), —(C₁-C₆alkylene)-S(O)₂R, —(C₁-C₆ alkylene)-S(O)₂—N(R)(R), —N(R)(R),—C(O)—N(R)(R), —N(R)—C(O)R, —N(R)—C(O)OR, —(C₁-C₆ alkylene)-N(R)—C(O)R,—N(R)S(O)₂R, and —P(O)(R)(R); wherein each of alkyl, alkenyl, alkynyl,alkoxy, heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl,heterocyclyl, and heterocyclylalkyl is independently substituted with0-5 occurrences of R^(a); or two R¹ or two R⁷ are taken together withthe carbon atoms to which they are attached form a cycloalkyl orheterocyclyl ring independently substituted with 0-5 occurrences ofR^(b);

each of R², R^(3a), R^(3b), R⁴, R^(8a) and R^(8b) is independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, hydroxyl, C₁-C₆heteroalkyl, and —N(R)(R); wherein each alkyl, alkoxy, and heteroalkylis optionally and independently substituted with 0-5 occurrences ofR^(a);

each of R⁵ and R⁹ is independently selected from hydrogen, C₁-C₆ alkyl,and C₁-C₆ heteroalkyl; wherein each alkyl and heteroalkyl is optionallyand independently substituted with 0-5 occurrences of R^(a);

each R⁶ is independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆alkoxy, halo, C₁-C₆ heteroalkyl, and —N(R)(R); wherein each alkyl,alkoxy, and heteroalkyl is optionally and independently substituted with0-5 occurrences of R^(a);

each R is independently selected from hydrogen, hydroxyl, halo, thiol,C₁-C₆ alkyl, C₁-C₆ thioalkyl, C₁-C₆ alkoxy, C₁-C₆ heteroalkyl,cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, andheterocyclylalkyl, wherein each of alkyl, thioalkyl, alkoxy,heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl,and heterocyclylalkyl is independently substituted with 0-5 occurrencesof R^(a), or two R together with the atom(s) to which they are attachedform a cycloalkyl or heterocyclyl ring independently substituted with0-5 occurrences of R^(b);

each R^(a) and each R^(b) is independently selected from C₁-C₆ alkyl,halo, hydroxyl, C₁-C₆ heteroalkyl, C₁-C₆ alkoxy, cycloalkyl,heterocyclyl, or cyano, wherein each of alkyl, heteroalkyl, alkoxy,cycloalkyl and heterocyclyl is independently substituted with 0-5occurrences of R′;

each R′ is independently selected from C₁-C₆ alkyl, C₁-C₆ heteroalkyl,halo, hydroxyl, cycloalkyl or cyano; or two R′ together with the atom(s)to which they are attached form a cycloalkyl or heterocyclyl ring;

m is 0, 1, or 2; and

n is 0, 1, 2, or 3.

In some embodiments, the compound has structural Formula (Ia):

or a pharmaceutically acceptable salt thereof,wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a),R^(8b), R⁹, m and n are as defined as for structural Formula (I).

In some embodiments, the compound has structural Formula (Ib):

or a pharmaceutically acceptable salt thereof,wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a),R^(8b), R⁹, m and n are as defined as for structural Formula (I).

In some embodiments of any of structural Formula I, (Ia) or (Ib), R¹ islocated at the 5-position. In some embodiments, R¹ is C₁-C₄ alkyloptionally substituted with 0-3 occurrences of R^(a). In someembodiments, m is 1 or 2. In some embodiments, m is 1. In someembodiments, m is 1; R¹ is located at the 5-position; and R¹ is C₁-C₄alkyl optionally substituted with 0-3 occurrences of R^(a). In someembodiments, R¹ is —CH₃.

In some embodiments of any of structural Formula I or (Ia), R² isselected from hydrogen, hydroxyl, halo and O—C₁-C₄ alkyl. In someembodiments, R² is selected from hydrogen, hydroxyl, fluoro, and —OCH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), eachof R^(3a), R^(3b), R^(8a) and R^(8b) is independently selected fromhydrogen and C₁-C₄ alkyl optionally substituted with 0-3 occurrences ofR^(a). In some embodiments, each of R^(3a), R^(3b), R^(8a) and R^(8b) isindependently selected from hydrogen and —CH₃. In some embodiments, atleast one pair of R^(3a) and R^(3b) or R^(8a) and R^(8b) issimultaneously hydrogen. In some embodiments, each of R^(3a), R^(3b),R^(8a) and R^(8b) is hydrogen.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁴ isselected from hydrogen, C₁-C₄ alkyl, and O—C₁-C₄ alkyl, wherein eachalkyl portion of R⁴ is optionally substituted with 0-3 occurrences ofR^(a). In some embodiments, R⁴ is selected from hydrogen, —CH₃ and—OCH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁵ isselected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3occurrences of R^(a). In some embodiments, R⁵ is selected from hydrogenand —CH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), eachR⁶ is independently selected from hydrogen, halo, and C₁-C₄ alkylsubstituted with 0-3 occurrences of R^(a). In some embodiments of any ofstructural Formulae I, (Ia) or (Ib), R⁶ is selected from hydrogen andC₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). Insome embodiments, each R⁶ is independently selected from hydrogen,chloro and —CH₃. In some embodiments, each R⁶ is independently selectedfrom hydrogen and —CH₃. In some embodiments, no more than one R⁶ isother than hydrogen.

In some embodiments of any of structural Formula I, (Ia) or (Ib), ring Ais a 6-membered monocyclic heteroaryl comprising at least one nitrogenring atom. In some embodiments, ring A is pyridine or pyrazine. In someembodiments, ring A is selected from

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁷ isheteroaryl optionally substituted with 0-3 occurrences of R^(b). In someembodiments, R⁷ is a 5-membered monocyclic heteroaryl optionallysubstituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ is a5-membered monocyclic nitrogen-containing heteroaryl (e.g., a 5-memberedmonocyclic heteroaryl comprising 1 or 2 nitrogen atoms) optionallysubstituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ ispyrazol-1-yl optionally substituted with 0-3 occurrences of R^(b). Insome embodiments, n is 1. In some embodiments, n is 1; and R⁷ ispyrazol-1-yl optionally substituted with 0-3 occurrences of R^(b). Insome embodiments, R⁷ is 4-fluoropyrazol-1-yl.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁹ isselected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3occurrences of R^(a). In some embodiments, R⁹ is hydrogen. In someembodiments, R⁹ is C₁-C₄ alkyl. In some embodiments, R⁵ and R⁹ are bothhydrogen.

In another aspect, the present invention features a compound having thestructural structural Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is selected from N, CH and C(halo);

X² is selected from N and CH;

X³ is selected from N and CH;

R¹² is selected from hydrogen, hydroxyl, halo and O—C₁-C₄ alkyl;

R¹⁴ is selected from hydrogen, —C₁-C₄ alkyl and —O—C₁-C₄ alkyl;

R¹⁵ is selected from hydrogen and —C₁-C₄ alkyl;

R¹⁶ is selected from hydrogen and —C₁-C₄ alkyl;

R¹⁷ is selected from hydrogen and halo; and

R¹⁹ is selected from hydrogen and —C₁-C₄ alkyl.

In some embodiments, —C₁-C₄ alkyl is optionally substituted. In someembodiments, —C₁-C₄ alkyl is substituted. In some embodiments, —C₁-C₄alkyl is unsubstituted.

In an embodiment, X¹ is N and R¹⁶ is —CH₃.

In some embodiments, the compound is a compound having the structuralFormula (IIa):

or a pharmaceutically acceptable salt thereof,wherein X¹, X², X³, R¹², R¹⁴, R¹⁵, R¹⁷, R¹⁶ and R¹⁹ are as defined asfor structural Formula (II).

In some embodiments, the compound is a compound having the structuralFormula (IIb):

or a pharmaceutically acceptable salt thereof,wherein X¹, X², X³, R¹², R¹⁴, R¹⁵, R¹⁷, R¹⁶ and R¹⁹ are as defined asfor structural Formula (II).

In some embodiments of any of structural Formula II, (IIa) or (IIb), X¹is N or CH. In some embodiments, X¹ is N.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X²is N or CH.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X³is N. In some embodiments of any of Formula II, (IIa) or (IIb), X³ isCH.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹²is selected from hydrogen, hydroxyl, fluoro and —OCH₃. In someembodiments, R¹² is hydrogen. In some embodiments, R¹² is hydroxyl.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁴is selected from hydrogen, —CH₃ and —OCH₃. In some embodiments, R¹⁴ ishydrogen. In some embodiments, R¹⁴ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹²is selected from hydrogen and hydroxyl; and R¹⁴ is selected fromhydrogen, —CH₃ and —OCH₃. In some embodiments, R¹² and R¹⁴ are bothhydrogen.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁵is H or —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁶is selected from hydrogen and —CH₃. In some embodiments, when X¹ is CH,R¹⁶ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X¹is N and R¹⁶ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁷is selected from hydrogen and fluoro. In some embodiments, R¹⁷ ishydrogen. In some embodiments, R¹⁷ is fluoro.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁹is H or —CH₃. In some embodiments, R¹⁹ is H.

In some embodiments, the present invention features a compound selectedfrom any compound in Table 1.

In another aspect, the present invention features a pharmaceuticalcomposition comprising a compound of structural Formula I, (Ia), (Ib),II, (IIa), or (IIb) described herein (e.g., a compound in Table 1) or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In another aspect, the present invention features a method forinhibiting RET activity in a cell or in a patient comprising the step ofcontacting the cell or administering to the patient a compound describedherein (e.g., a compound in Table 1) or a pharmaceutically acceptablesalt thereof, or a pharmaceutical composition thereof.

In another aspect, the present invention features a method for treatinga subject suffering from a condition mediated by aberrant RET activity,comprising administering to the subject a therapeutically effectiveamount of a compound described herein (e.g., a compound in Table 1) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof.

In another aspect, the present invention features a method for treatinga subject who has developed resistance to a cancer treatment, comprisingadministering to the subject a therapeutically effective amount of acompound described herein (e.g., a compound in Table 1) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof. In some embodiments, the subject has developedresistance to a wild-type RET inhibitor.

TABLE 1 Exemplary Compounds of the Invention. Compound Structure 100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

Pharmaceutically acceptable salts of these compounds are alsocontemplated for the uses described herein.

“Pharmaceutically acceptable salt” refers to any salt of a compound ofthe invention which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Pharmaceuticallyacceptable salts may be derived from a variety of organic and inorganiccounter-ions well known in the art and include. Such salts include: (1)acid addition salts formed with organic or inorganic acids such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic,acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like. Pharmaceutically acceptablesalts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, besylate, acetate, maleate, oxalate and the like.

Pharmaceutical Compositions

Pharmaceutical compositions of the invention comprise one or morecompounds of the invention and one or more physiologically orpharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to a pharmaceutically-acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition or component thereof. Each carriermust be “acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The compositions of the invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. In some embodiments, the compositions of the invention areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the compositions of this invention may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tween, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs. Topical application for the lower intestinal tract canbe effected in a rectal suppository formulation (see above) or in asuitable enema formulation. Topically-transdermal patches may also beused.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration.

Dosages

Toxicity and therapeutic efficacy of compounds of the invention,including pharmaceutically acceptable salts and deuterated variants, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals. The LD₅₀ is the dose lethal to 50% of thepopulation. The ED₅₀ is the dose therapeutically effective in 50% of thepopulation. The dose ratio between toxic and therapeutic effects(LD₅₀/ED₅₀) is the therapeutic index. Compounds that exhibit largetherapeutic indexes are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Treatment

RET fusions have been implicated in several types of cancers. Generally,these RET fusions have a RET kinase domain that is the same as inwild-type RET; therefore, as used herein, any RET protein with the samekinase domain as wild-type RET will be referred to as “wild-type RET”unless noted otherwise. Mutations can occur at least in the RETextracellular and kinase domains. Mutations can occur in the RET kinasedomain, leading to resistant mutants of RET.

The activity of exemplary compounds that are approved or in developmentfor RET-related conditions is shown below. As shown, the compounds areactive against the wild-type RET, but are much less active against themutated forms (“wild-type RET inhibitors”).

TABLE 2 RET wt RET V804L Biochemical Biochemical Compound IC₅₀ (nM) IC₅₀(nM) Cabozantinib 46 219 Vandetanib 1.2 902 Sorafenib 7.9 95.2Regorafenib 5.1 29.8

The invention provides compounds that inhibit both wild-type RET andmutants of RET, e.g., mutants of RET that are resistant to currentstandard of care treatments (“RET resistant mutants”). In addition, thecompounds of the invention can be selective for wild-type RET, overother kinases, thus leading to reduced toxicities associated withinhibiting other kinases. In one aspect, compounds of the invention areselective for RET over KDR. In one aspect, compounds of the invention donot cause adverse effects such as hypertension, arterial thrombosis, andhemorrhage.

Mutations can be predicted using structural biology and computationalanalyses, as well as by examining codon sequences in which a sequencechange gives rise to a codon for a different amino acid. Using suchmethods, RET resistant mutants are predicted to have point mutations atthe 804 gatekeeper residue in the RET protein and/or at residues at ornear the gatekeeper residue. In some embodiments, the mutation may be atone or more of the 804, 806, 810, 865, 870, 891, and 918 residues.Specific examples of RET resistant mutants include: V804L, V804M, V804E,Y806C, Y806S, Y806H, Y806N, G810R, G810S, L865V, L870F, S891A and M918Tmutants.

Mutations occurring from administration of a particular inhibitor (e.g.,a known RET wild-type inhibitor) can be determined experimentally byexposing cells to a mutation-promoting agent, such as ENU. The cells arewashed, then plated with increasing concentrations (2-100× proliferationIC₅₀) of the compound of choice. The wells with cellular outgrowth arethen collected after 3-4 weeks. The RET kinase domain is then sequencedto identify resistance mutations (i.e., altered forms of the RET proteinthat retain enzymatic activity). Resistance can be confirmed by exposingthese cells with the compound of choice. Resistant mutants that havebeen identified experimentally include the V804L, V804E, V804M, andY806H mutants. In some embodiments, the mutation is a substitution ofcysteine (C609, C611, C618, C620, C630, and C634) in the RETextracellular domain for any other amino acid. In some embodiments, theRET cysteine variants (affecting C609, C611, C618, and C620) are the“Janus mutations.” In some embodiments, RET mutations include: RETC634W, RET M918T, V804L, V804E, V804M, V804L, Y806C, Y806S, Y806N,Y806H, G810R, G810S, L865V, L870F, and S891A mutants.

Because of their activity against wild-type RET and mutant RET, thecompounds described herein can be used to treat a patient with acondition associated with aberrant RET activity. The compounds describedherein can provide treatments for irritable bowel syndrome (IBS),proliferative diseases, and any other conditions related to aberant RETactivity. The compounds can be used to treat irritable bowel syndrome.The compounds can be used to treat various cancers. In some embodiments,the cancer is selected from papillary thyroid carcinoma (PTC), medullarythyroid cancer (MTC), pheochromocytoma (PC), pancreatic ductaladenocarcinoma, multiple endocrine neoplasia (MEN2A and MEN2B),metastatic breast cancer, testicular cancer, small cell lung cancer,non-small cell lung cancer, chronic myelomonocytic leukemia, colorectalcancer, ovarian cancer, and cancers of the salivary gland. In someembodiments, the cancer is a solid tumor. In some embodiments, thecondition associated with aberrant RET activity is a thyroid cancer(e.g., papillary thyroid carcinoma, thyroid adenocarcinoma, or MTC,e.g., familial MTC), lung cancer (e.g., lung adenocarcinoma, small-celllung carcinoma, or non-small cell lung carcinoma), breast cancer (e.g.,estrogen receptor-positive tumors and endocrine-resistant tumors e.g.,resistant to oestrogen modulators such as tamoxifen, agents that blockoestrogen biosynthesis such as aromatase inhibitors, and oestrogenreceptor antagonists such as fulvestrant), pancreatic cancer (e.g.,carcinoma of the pancreas or pancreatic ductal carcinoma),haematopoietic cancer, e.g., a leukemia (e.g., chronic myelomonocyticleukemia or acute myeloid leukemia), colon cancer (e.g., coloncarcinoma), melanoma (e.g., cutaneous or desmoplastic malignantmelanomas), prostate cancer, renal cancer (e.g., renal cell carcinoma),and head and neck tumors, neuroblastoma, ganglioneuroma (e.g.,ganglioneuroma of the mouth or gut), colon cancer (e.g., sporadic coloncancers), MEN2A (multiple endocrine neoplasia type 2A), or MEN2B(multiple endocrine neoplasia type 2B). In one aspect, the MEN2A ischaracterized by MTC and includes adrenal tumor pheochromocytoma.Substitutions of cysteines in RET are found in subjects with MEN2A andalso frequent in FMTC. RET extracellular domain exon 8 mutations, suchas G533C) or the RET intracellular domain (residues E768, L790, Y791,V804, and S891) are associated with FMTC or MEN2A. Substitutions in theRET kinase domain, Met918 to Thr (M918T) or A883F are found in subjectswith MEN2B. RET M918T and RET A883F are also found in sporadic MTC.

The compounds can also be used to treat a patient who has developedresistance to a wild-type RET inhibitor, or a patient with a particularRET mutant. The method includes the step of administering a compound orcomposition of the invention that is active against one or more RETresistant mutants. In certain embodiments, the RET resistant mutant isselected from V804L, V804M, V804E, Y806C, Y806S, Y806N, Y806H, G810R,G810S, L865V, L870F, S891A and M918T. By “active” is meant that acompound has an IC₅₀ of less than 1 μM, 500 nM, 250 nM, 100 nM, 75 nM,50 nM, 25 nM, 10 nM, or 5 nM when measured in a biochemical assay,against at least one resistant mutant.

The compounds may also be used to treat a subject having a RET-alteredcell, cancer, gene, or gene product. The RET alteration may be, e.g., apoint mutation, insertion, deletion, amplification, or fusion, or acombination thereof.

The compounds may also be used to treat a subject having a RET-alteredcell, cancer, gene, or gene product comprising a RET alterationdescribed in Table 3 or Table 4 herein. In some embodiments, the subjecthas a fusion between RET and a RET fusion partner listed in Table 3,e.g., comprises a fusion protein that comprises RET or a fragmentthereof and a protein of Table 3 or fragment thereof. In someembodiments, the fusion partner is N-terminal or C-terminal of RET. Insome embodiments, the subject has an alteration at a position in RETthat is described in Table 4. In some embodiments, a subset of thesubject's cells, e.g., a subset of the subject's tumor cells, comprisethe RET alteration. In some embodiments, a subset of the subject'scells, e.g., a subset of the subject's tumor cells, are RET-altered. Insome embodiments, the subject has a cancer listed in Table 3, e.g., thesubject has both a RET mutation and a cancer listed in Table 3.

TABLE 3 RET fusions RET fusion partner Exemplary cancers in which thefusion is found BCR Chronic Myelomonocytic Leukemia (CMML) CLIP 1Adenocarcinoma KIFSB NSCLC, Ovarian Cancer, Spitzoid Neoplasm; LungAdenocarcinoma, Adenosquamous Carcinomas CCDC6 NSCLC, Colon Cancer,Papillary Thyroid Cancer; Adenocarcinoma; Lung Adenocarcinoma;Metastatic Colorectal Cancer; Adenosquamous Carcinoma, Metastaticpapillary thyroid cancer PTClex9 Metastatic papillary thyroid cancerNCOA4 Papillary Thyroid Cancer, NSCLC, Colon Cancer, Salivary GlandCancer, Metastatic Colorectal Cancer; Lung Adenocarcinoma, AdenosquamousCarcinomas; Diffuse Sclerosing Variant of Papillary Thyroid CancerTRIM33 NSCLC, Papillary Thyroid Cancer ERC1 Papillary Thyroid Cancer,Breast Cancer FGFRIOP CMML, Primary Myelofibrosis with secondary AcuteMyeloid Leukemia MBD1 Papillary Thyroid Cancer RAB61P2 Papillary ThyroidCancer PRKAR1A Papillary Thyroid Cancer TRIM24 Papillary Thyroid CancerKTN1 Papillary Thyroid Cancer GOLGA5 Papillary Thyroid Cancer, SpitzoidNeoplasms HOOK3 Papillary Thyroid Cancer KIAA1468 Papillary ThyroidCancer, Lung Adenocarcinoma TRIM27 Papillary Thyroid Cancer AKAP13Papillary Thyroid Cancer FKBP15 Papillary Thyroid Cancer SPECC1LPapillary Thyroid Cancer, Thyroid Gland Carcinoma TBL1XR1 PapillaryThyroid Cancer, Thyroid Gland Carcinoma CEP55 Diffuse Gastric CancerCUX1 Lung Adenocarcinoma ACBD5 Papillary Thyroid Carcinoma MYH13Medullary Thyroid Carcinoma PIBF1 Bronchiolus Lung Cell CarcinomaKIAA1217 Papillary Thyroid Cancer, Lung Adenocarcinoma, NSCLC MPRIPNSCLC

TABLE 4 RET mutations Amino acid position 2 Amino acid position 665(e.g., H665Q) Amino acid position 3 Amino acid position 666 (e.g.,K666E, K666M, or K666N) Amino acid position 4 Amino acid position 686(e.g., S686N) Amino acid position 5 Amino acid position 691 (e.g.,G691S) Amino acid position 6 Amino acid position 694 (e.g., R694Q) Aminoacid position 7 Amino acid position 700 (e.g., M700L) Amino acidposition 8 Amino acid position 706 (e.g., V706M or V706A) Amino acidposition 11 Amino acid position 713 splice variant (e.g., E713K) Aminoacid position 12 Amino acid position 736 (e.g., G736R) Amino acidposition 13 Amino acid position 748 (e.g., G748C) Amino acid position 20Amino acid position 750 (e.g., A750P) Amino acid position 32 (e.g.,S32L) Amino acid position 765 (e.g., S765P) Amino acid position 34(e.g., D34S) Amino acid position 766 (e.g., P766S or P766M6) Amino acidposition 40 (e.g., L40P) Amino acid position 768 (e.g., E768Q or E768D)Amino acid position 64 (e.g., P64L) Amino acid position 769 (e.g.,L769L) Amino acid position 67 (e.g., R67H) Amino acid position 770(e.g., R770Q) Amino acid position 114 (e.g., R114H) Amino acid position771 (e.g., D771N) Amino acid position 136 (e.g., glutamic Amino acidposition 777 (e.g., N777S) acid to stop codon) Amino acid position 145(e.g., V145G) Amino acid position 778 (e.g., V778I) Amino acid position180 (e.g., arginine to Amino acid position 781 (e.g., Q781R) stop codon)Amino acid position 200 Amino acid position 790 (e.g., L790F) Amino acidposition 292 (e.g., V292M) Amino acid position 791 (e.g., Y791F orY791N) Amino acid position 294 Amino acid position 802 Amino acidposition 321 (e.g., G321R) Amino acid position 804 (e.g., V804L, V804M,V804M*,orV804E) Amino acid position 330 (e.g., R330Q) Amino acidposition 805 (e.g., E805K) Amino acid position 338 (e.g., T338I) Aminoacid position 806 (e.g., E806C, Y806E, Y806F, Y806S, Y806G, or Y806C)Amino acid position 360 (e.g., R360W) Amino acid position 818 (e.g.,E818K) Amino acid position 373 (e.g., alanine to Amino acid position 819(e.g., S819I) frameshift) Amino acid position 393 (e.g., F393L) Aminoacid position 823 (e.g., G823E) Amino acid position 432 Amino acidposition 826 (e.g., Y826M) Δ Amino acid residues 505-506 (6-Base Aminoacid position 833 (e.g., R833C) Pair In-Frame Germline Deletion in Exon7) Amino acid position 510 (e.g., A510V) Amino acid position 841 (e.g.,P841L or P841P) Amino acid position 511 (e.g., E51IK) Amino acidposition 843 (e.g., E843D) Amino acid position 513 (e.g., A513D) Aminoacid position 844 (e.g., R844W, R844Q, or R844L) Amino acid position 515(e.g., C515S, Amino acid position 848 (e.g., M848T) C515W) Amino acidposition 525 (e.g., R525W) Amino acid position 852 (e.g., 1852M) Aminoacid position 531 (e.g., C531R, or Amino acid position 866 (e.g., A866W)9 base pair duplication) Amino acid position 532 (e.g., Amino acidposition 873 (e.g., R873W) duplication) Amino acid position 533 (e.g.,G533C or G533S) Amino acid position 876 (e.g., A876V) Amino acidposition 550 (e.g., G550E) Amino acid position 881 (e.g., L881V) Aminoacid position 591 (e.g., V591I) Amino acid position 882 Amino acidposition 593 (e.g., G593E) Amino acid position 883 (e.g., A883F, A883S,A883T, or A883T*) Amino acid position 600 (e.g., R600Q) Amino acidposition 884 (e.g., E884K) Amino acid position 602 (e.g., I602V) Aminoacid position 886 (e.g., R886W) Amino acid position 603 (e.g., K603Q orAmino acid position 891 (e.g., S891A) K603E2) Amino acid position 606(e.g., Y606C) Amino acid position 897 (e.g., R897Q) Amino acid position609 (e.g., C609Y, Amino acid position 898 (e.g., D898V) C609S, C609G,C609R, C609F, or C609W) Amino acid position 611 (e.g., C611R, Amino acidposition 901 (e.g., E901K) C611S, C611G, C611Y, C611F, or C611W) Aminoacid position 618 (e.g., C618S, Amino acid position 904 (e.g., S904F orS904C2) C618Y, C618R, C618Y, C618G, C618F, C618W) Amino acid position619 (e.g., F619F) Amino acid position 907 (e.g., K907E or K907M) Aminoacid position 620 (e.g., C620S, Amino acid position 908 (e.g., R908K)C620W, C620R, C620G, C620L, C620Y, C620F) Amino acid position 623 (e.g.,E623K) Amino acid position 911 (e.g., G911D) Amino acid position 624(e.g., D624N) Amino acid position 912 (e.g., R912P, R912Q) Amino acidposition 630 (e.g., C630A, Amino acid position 918 (e.g., M918T, M918V,or C630R, C630S, C630Y, or C630F) M918L6) Amino acid position 631 (e.g.,D631N, Amino acid position 919 (e.g., A919V) D631Y, D631A, D631G, D631V,or D631E) Amino acid position 632 (e.g., E632K or Amino acid position921 (e.g., E921K) E632G5) Δ Amino acid residues 632-633 (6-Base Aminoacid position 922 (e.g., S922P or S922Y) Pair In-Frame Germline Deletionin Exon 11) Amino acid position 633 (e.g., 9 base pair Amino acidposition 930 (e.g., T930M) duplication) Amino acid position 634 (e.g.,C634W, Amino acid position 961 (e.g., F961L) C634Y, C634S, C634R, C634F,C634G, C634L, C634A, or C634T, or an insertion ELCR2, or a 12 base pairduplication) Amino acid position 635 (e.g., R635G) Amino acid position972 (e.g., R972G) Amino acid position 636 (e.g., T636P or Amino acidposition 982 (e.g., R982C) T636M4) Amino acid position 640 (e.g., A640G)Amino acid position 1009 (e.g., M 1009V) Amino acid position 641 (e.g.,A641S or Amino acid position 1017 (e.g., D1017N) A641T8) Amino acidposition 648 (e.g., V6481) Amino acid position 1041 (e.g., V1041G) Aminoacid position 649 (e.g., S649L) Amino acid position 1064 (e.g., M1064T)Amino acid position 664 (e.g., A664D) RET + 3

RET has two primary protein and mRNA isoforms, named RET51 and RETS. Inembodiments, RET has a sequence of isoform RET51 as shown below as SEQID NO: 1. The kinase domain, corresponding to amino acids 724-1016 ofSEQ ID NO: 1, is highlighted. Unless otherwise indicated, the amino acidpositions described herein refer to the numbering of RET51 shown below.

SEQ ID NO: 1:         10         20         30         40         50MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL        60         70         80         90        100LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS       110        120        130        140        150LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF       160        170        180        190        200NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI       210        220        230        240        250SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV AVCTVHAGAR       260        270        280        290        300EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TVVATLRVFD       310        320        330        340        350ADVVPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT       360        370        380        390        400VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV       410        420        430        440        450SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS       460        470        480        490        500TLGVVTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL       510        520        530        540        550LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG       560        570        580        590        600ITRNFSTCSP STKTCPDGHC DVVETQDINI CPQDCLRGSI VGGHEPGEPR       610        620        630        640        650GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV       660        670        680        690        700LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM       710        720        730        740        750ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKVVK ATAFHLKGRA       760        770        780        790        800GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL       810        820        830        840        850LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD       860        870        880        890        900LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY       910        920        930        940        950EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN       960        970        980        990       1000PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD      1010       1020       1030       1040       1050ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR      1060       1070       1080       1090       1100ALPSTWIENK LYGMSDPNWP GESPVPLTRA DGTNTGFPRY PNDSVYANWM       1110LSPSAAKLMD TFDS

In embodiments, RET has a sequence of isoform RETS as shown below in SEQID NO: 2 (wherein the kinase domain is highlighted)

SEQ ID NO: 2:         10         20         30         40         50MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL        60         70         80         90        100LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS       110        120        130        140        150LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF        160        170       180        190        200NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI       210        220        230        240        250SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV AVCTVHAGAR       260        270        280        290        300EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TVVATLRVFD       310        320        330        340        350ADVVPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT       360        370        380        390        400VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV       410        420        430        440        450SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS       460        470        480        490        500TLGVVTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL       510        520        530        540        550LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG       560        570        580        590        600ITRNFSTCSP STKTCPDGHC DVVETQDINI CPQDCLRGSI VGGHEPGEPR       610        620        630        640        650GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV       660        670        680        690        700LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM        710       720        730        740        750ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKVVK ATAFHLKGRA       760        770        780        790        800GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL       810        820        830        840        850LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD       860        870        880        890        900LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY       910        920        930        940        950EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN       960        970        980        990       1000PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD      1010       1020       1030       1040       1050ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR      1060       1070 ALPSTWIENK LYGRISHAFT RF

In embodiments, RET51 is encoded by a nucleic acid having the sequenceof

SEQ ID NO: 3:    1agtcccgcga ccgaagcagg gcgcgcagca gcgctgagtg ccccggaacg tgcgtcgcgc   61ccccagtgtc cgtcgcgtcc gccgcgcccc gggcggggat ggggcggcca gactgagcgc  121cgcacccgcc atccagaccc gccggcccta gccgcagtcc ctccagccgt ggccccagcg  181cgcacgggcg atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct  241gctgctgccg ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg  301ggagaagctg tatgtggacc aggcggccgg cacgcccttg ctgtacgtcc atgccctgcg  361ggacgcccct gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg  421cacacggctg catgagaaca actggatctg catccaggag gacaccggcc tcctctacct  481taaccggagc ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc  541cctgctcacc gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg  601ccagtggcca ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg  661cagctccctc aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg  721ggagaaccga cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg  781ccccaacatc agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc  841cccggacagc ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta  901cgagctggtg gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc  961cttcccggtg accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga 1021caccgccagc gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg 1081tgtcttcgat gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac 1141gctgctcccc ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga 1201gacctcggtc caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt 1261tctcaaccgg aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa 1321tgactcagac ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt 1381gctgccggtc agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg 1441ccgatttgcc cagatcggga aagtctgtgt ggaaaactgc caggcattca gtggcatcaa 1501cgtccagtac aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc 1561agccgaggac acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa 1621gtgtgccgaa cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca 1681ggcccagctg cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct 1741gtcctgtgca gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc 1801aacaggcagg tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac 1861ctgctctccc agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga 1921catcaacatt tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg 1981ggagccccgg gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa 2041gtgcttctgc gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt 2101gatcgcagcc gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat 2161ccactgctac cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg 2221gaggcccgcc caggccttcc cggtcagcta ctcctcttcc ggtgcccgcc ggccctcgct 2281ggactccatg gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg 2341ggaattccct cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa 2401agtggtcaag gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt 2461gaagatgctg aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa 2521cgtcctgaag caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga 2581tggcccgctc ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg 2641cgagagccgc aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc 2701cctggaccac ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca 2761gatctcacag gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc 2821cagaaacatc ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg 2881agatgtttat gaagaggatt cctacgtgaa gaggagccag ggtcggattc cagttaaatg 2941gatggcaatt gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt 3001tggtgtcctg ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc 3061tgagcggctc ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag 3121cgaggagatg taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt 3181gtttgcggac atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga 3241ccttgcggcg tccactccat ctgactccct gatttatgac gacggcctct cagaggagga 3301gacaccgctg gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat 3361tgaaaacaaa ctctatggca tgtcagaccc gaactggcct ggagagagtc ctgtaccact 3421cacgagagct gatggcacta acactgggtt tccaagatat ccaaatgata gtgtatatgc 3481taactggatg ctttcaccct cagcggcaaa attaatggac acgtttgata gttaacattt 3541ctttgtgaaa ggtaatggac tcacaagggg aagaaacatg ctgagaatgg aaagtctacc 3601ggccctttct ttgtgaacgt cacattggcc gagccgtgtt cagttcccag gtggcagact 3661cgtttttggt agtttgtttt aacttccaag gtggttttac ttctgatagc cggtgatttt 3721ccctcctagc agacatgcca caccgggtaa gagctctgag tcttagtggt taagcattcc 3781tttctcttca gtgcccagca gcacccagtg ttggtctgtg tccatcagtg accaccaaca 3841ttctgtgttc acatgtgtgg gtccaacact tactacctgg tgtatgaaat tggacctgaa 3901ctgttggatt tttctagttg ccgccaaaca aggcaaaaaa atttaaacat gaagcacaca 3961cacaaaaaag gcagtaggaa aaatgctggc cctgatgacc tgtccttatt cagaatgaga 4021gactgcgggg ggggcctggg ggtagtgtca atgcccctcc agggctggag gggaagaggg 4081gccccgagga tgggcctggg ctcagcattc gagatcttga gaatgatttt tttttaatca 4141tgcaaccttt ccttaggaag acatttggtt ttcatcatga ttaagatgat tcctagattt 4201agcacaatgg agagattcca tgccatcttt actatgtgga tggtggtatc agggaagagg 4261gctcacaaga cacatttgtc ccccgggccc accacatcat cctcacgtgt tcggtactga 4321gcagccacta cccctgatga gaacagtatg aagaaagggg gctgttggag tcccagaatt 4381gctgacagca gaggctttgc tgctgtgaat cccacctgcc accagcctgc agcacacccc 4441acagccaagt agaggcgaaa gcagtggctc atcctacctg ttaggagcag gtagggcttg 4501tactcacttt aatttgaatc ttatcaactt actcataaag ggacaggcta gctagctgtg 4561ttagaagtag caatgacaat gaccaaggac tgctacacct ctgattacaa ttctgatgtg 4621aaaaagatgg tgtttggctc ttatagagcc tgtgtgaaag gcccatggat cagctcttcc 4681tgtgtttgta atttaatgct gctacaagat gtttctgttt cttagattct gaccatgact 4741cataagcttc ttgtcattct tcattgcttg tttgtggtca cagatgcaca acactcctcc 4801agtcttgtgg gggcagcttt tgggaagtct cagcagctct tctggctgtg ttgtcagcac 4861tgtaacttcg cagaaaagag tcggattacc aaaacactgc ctgctcttca gacttaaagc 4921actgatagga cttaaaatag tctcattcaa atactgtatt ttatataggc atttcacaaa 4981aacagcaaaa ttgtggcatt ttgtgaggcc aaggcttgga tgcgtgtgta atagagcctt 5041gtggtgtgtg cgcacacacc cagagggaga gtttgaaaaa tgcttattgg acacgtaacc 5101tggctctaat ttgggctgtt tttcagatac actgtgataa gttcttttac aaatatctat 5161agacatggta aacttttggt tttcagatat gcttaatgat agtcttacta aatgcagaaa 5221taagaataaa ctttctcaaa ttattaaaaa tgcctacaca gtaagtgtga attgctgcaa 5281caggtttgtt ctcaggaggg taagaactcc aggtctaaac agctgaccca gtgatgggga 5341atttatcctt gaccaattta tccttgacca ataacctaat tgtctattcc tgagttataa 5401aagtccccat ccttattagc tctactggaa ttttcataca cgtaaatgca gaagttacta 5461agtattaagt attactgagt attaagtagt aatctgtcag ttattaaaat ttgtaaaatc 5521tatttatgaa aggtcattaa accagatcat gttccttttt ttgtaatcaa ggtgactaag 5581aaaatcagtt gtgtaaataa aatcatgtat cataaaaaaa aaaaaaaaa //

In embodiments, RETS is encoded by a nucleic acid having the sequence of

SEQ ID NO: 4:    1agtcccgcga ccgaagcagg gcgcgcagca gcgctgagtg ccccggaacg tgcgtcgcgc   61ccccagtgtc cgtcgcgtcc gccgcgcccc gggcggggat ggggcggcca gactgagcgc  121cgcacccgcc atccagaccc gccggcccta gccgcagtcc ctccagccgt ggccccagcg  181cgcacgggcg atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct  241gctgctgccg ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg  301ggagaagctg tatgtggacc aggcggccgg cacgcccttg ctgtacgtcc atgccctgcg  361ggacgcccct gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg  421cacacggctg catgagaaca actggatctg catccaggag gacaccggcc tcctctacct  481taaccggagc ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc  541cctgctcacc gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg  601ccagtggcca ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg  661cagctccctc aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg  721ggagaaccga cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg  781ccccaacatc agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc  841cccggacagc ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta  901cgagctggtg gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc  961cttcccggtg accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga 1021caccgccagc gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg 1081tgtcttcgat gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac 1141gctgctcccc ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga 1201gacctcggtc caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt 1261tctcaaccgg aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa 1321tgactcagac ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt 1381gctgccggtc agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg 1441ccgatttgcc cagatcggga aagtctgtgt ggaaaactgc caggcattca gtggcatcaa 1501cgtccagtac aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc 1561agccgaggac acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa 1621gtgtgccgaa cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca 1681ggcccagctg cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct 1741gtcctgtgca gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc 1801aacaggcagg tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac 1861ctgctctccc agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga 1921catcaacatt tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg 1981ggagccccgg gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa 2041gtgcttctgc gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt 2101gatcgcagcc gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat 2161ccactgctac cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg 2221gaggcccgcc caggccttcc cggtcagcta ctcctcttcc ggtgcccgcc ggccctcgct 2281ggactccatg gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg 2341ggaattccct cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa 2401agtggtcaag gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt 2461gaagatgctg aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa 2521cgtcctgaag caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga 2581tggcccgctc ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg 2641cgagagccgc aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc 2701cctggaccac ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca 2761gatctcacag gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc 2821cagaaacatc ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg 2881agatgtttat gaagaggatt cctacgtgaa gaggagccag ggtcggattc cagttaaatg 2941gatggcaatt gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt 3001tggtgtcctg ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc 3061tgagcggctc ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag 3121cgaggagatg taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt 3181gtttgcggac atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga 3241ccttgcggcg tccactccat ctgactccct gatttatgac gacggcctct cagaggagga 3301gacaccgctg gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat 3361tgaaaacaaa ctctatggta gaatttccca tgcatttact agattctagc accgctgtcc 3421cctctgcact atccttcctc tctgtgatgc tttttaaaaa tgtttctggt ctgaacaaaa 3481ccaaagtctg ctctgaacct ttttatttgt aaatgtctga ctttgcatcc agtttacatt 3541taggcattat tgcaactatg tttttctaaa aggaagtgaa aataagtgta attaccacat 3601tgcccagcaa cttaggatgg tagaggaaaa aacagatcag ggcggaactc tcaggggaga 3661ccaagaacag gttgaataag gcgcttctgg ggtgggaatc aagtcatagt acttctactt 3721taactaagtg gataaatata caaatctggg gaggtattca gttgagaaag gagccaccag 3781caccactcag cctgcactgg gagcacagcc aggttccccc agacccctcc tgggcaggca 3841ggtgcctctc agaggccacc cggcactggc gagcagccac tggccaagcc tcagccccag 3901tcccagccac atgtcctcca tcaggggtag cgaggttgca ggagctggct ggccctggga 3961ggacgcaccc ccactgctgt tttcacatcc tttcccttac ccaccttcag gacggttgtc 4021acttatgaag tcagtgctaa agctggagca gttgcttttt gaaagaacat ggtctgtggt 4081gctgtggtct tacaatggac agtaaatatg gttcttgcca aaactccttc ttttgtcttt 4141gattaaatac tagaaattta aaaaaaaaaa aaaa //

The compounds and compositions described herein can be administeredalone or in combination with other compounds, including otherRET-modulating compounds, or other therapeutic agents. In someembodiments, the compound or composition of the invention may beadministered in combination with one or more compounds selected fromCabozantinib (COMETRIQ), Vandetanib (CALPRESA), Sorafenib (NEXAVAR),Sunitinib (SUTENT), Regorafenib (STAVARGA), Ponatinib (ICLUSIG),Bevacizumab (AVASTIN), Crizotinib (XALKORI), or Gefitinib (IRESSA). Thecompound or composition of the invention may be administeredsimultaneously or sequentially with the other therapeutic agent by thesame of different routes of administration. The compound of theinvention may be included in a single formulation with the othertherapeutic agent or in separate formulations.

EXAMPLES

The following examples are intended to be illustrative, and are notmeant in any way to be limiting.

Synthesis

Compounds of the invention, including salts and N-oxides thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, such as those inthe Schemes below. The reactions for preparing compounds of theinvention can be carried out in suitable solvents which can be readilyselected by one of skill in the art of organic synthesis. Suitablesolvents can be substantially non-reactive with the starting materials(reactants), the intermediates, or products at the temperatures at whichthe reactions are carried out, e.g., temperatures which can range fromthe solvent's freezing temperature to the solvent's boiling temperature.A given reaction can be carried out in one solvent or a mixture of morethan one solvent. Depending on the particular reaction step, suitablesolvents for a particular reaction step can be selected by the skilledartisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in Wuts and Greene,Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: NewJersey, (2006), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹Hor ¹³C), infrared (IR) spectroscopy, spectrophotometry (e.g.,UV-visible), mass spectrometry (MS), or by chromatographic methods suchas high performance liquid chromatography (HPLC) or thin layerchromatography (TLC). Analytical instruments and methods for compoundcharacterization:

LC-MS:

Unless otherwise indicated, all liquid chromatography-mass spectrometry(LC-MS) data (sample analyzed for purity and identity) were obtainedwith an Agilent model-1260 LC system using an Agilent model 6120 massspectrometer utilizing ES-API ionization fitted with an Agilent Poroshel120 (EC-C18, 2.7 um particle size, 3.0×50 mm dimensions) reverse-phasecolumn at 22.4 degrees Celsius. The mobile phase consisted of a mixtureof solvent 0.1% formic acid in water and 0.1% formic acid inacetonitrile. A constant gradient from 95% aqueous/5% organic to 5%aqueous/95% organic mobile phase over the course of 4 minutes wasutilized. The flow rate was constant at 1 mL/min.

Prep LC-MS:

Preparative HPLC was performed on a Shimadzu Discovery VP® Preparativesystem fitted with a Luna 5u C18(2) 100A, AXIA packed, 250×21.2 mmreverse-phase column at 22.4 degrees Celsius. The mobile phase consistedof a mixture of solvent 0.1% formic acid in water and 0.1% formic acidin acetonitrile. A constant gradient from 95% aqueous/5% organic to 5%aqueous/95% organic mobile phase over the course of 25 minutes wasutilized. The flow rate was constant at 20 mL/min. Reactions carried outin a microwave were done so in a Biotage Initiator microwave unit.

Silica Gel Chromatography:

Silica gel chromatography was performed on either a Teledyne IscoCombiFlash® Rf unit or a Biotage® Isolera Four unit.

Proton NMR:

Unless otherwise indicated, all ¹H NMR spectra were obtained with aVarian 400 MHz Unity Inova 400 MHz NMR instrument (acquisition time=3.5seconds with a 1 second delay; 16 to 64 scans). Where characterized, allprotons were reported in DMSO-d6 solvent as parts-per million (ppm) withrespect to residual DMSO (2.50 ppm).

The below Schemes are meant to provide general guidance in connectionwith preparing the compounds of the invention. One skilled in the artwould understand that the preparations shown in the Schemes can bemodified or optimized using general knowledge of organic chemistry toprepare various compounds of the invention.

Synthetic Protocol 1:

An aryl dihalide can be treated with an organolithium or organomagnesiumhalide reagent, such as n-BuLi or i-PrMgCl, and the arylmetal reagentcan then undergo addition to an ester substituted cyclobutanone (eithercommercially available or prepared as described in “Synthesis of KetoneIntermediates” in Example 6). The remaining halide can then undergo acoupling reaction with an arylamine under SnAr conditions ormetal-catalyzed coupling conditions to give a tricyclic esterintermediate. The ester can then be hydrolyzed under basic conditions togive an acid, which can then undergo an amide coupling reaction with anamine (either commercially available or prepared as described in“Synthesis of Amine Intermediates” in Example 8). The amides areexamples of RET inhibitors described herein, but can also be furthermodified. For example, the tertiary alcohol can be treated with afluorinating reagent such as DAST to give a mixture of deoxyfluorinatedproducts which are also examples of RET inhibitors.

Synthetic Protocol 2:

A substituted cycloalkyl iodide (either commercially available orprepared as described in “Synthesis of Iodide Intermediates”) is treatedwith activated zinc. The zinc could be activated by a variety methods,including but not limited to the method of Reike or treatment withTMS-Cl and 1,2-dibromoethane. The cycloalkyl zinc reagent can then becoupled to a heteroaryl dihalide with palladium catalysis under Negishicoupling conditions. The resulting carbon-carbon can form to give amixture of diastereomers, which can be separated by chromatography orother separation techniques at this stage. Alternatively thediastereomers can be used as a mixture in the subsequent transformationsand separated at a different stage. The remaining heteroaryl halide canthen undergo displacement with an aryl amine under either SnArconditions or palladium mediated coupling conditions. The tricycliccarboxylic ester can then be hydrolyzed under acidic or basic conditionsto provide a carboxylic acid intermediate. The carboxylic acidintermediate can then be coupled to a variety of amines, such as thosedescribed below under the heading “Synthesis of Amine Intermediates” inExample 8, to provide the amide final product.

Synthetic Protocol 3:

A heteroaryl dihalide can be coupled to an amino pyrazole undernucleophilic aromatic substitution reaction conditions using a base suchas diisopropylethylamine (DIPEA) or triethylamine (TEA) in a polarsolvent to provide the bicyclic ring system. The bicyclic heteroarylhalide can then be coupled to an excess of alkyl zinc reagent (typically2 equivalents)(M=Zn) via a palladium-mediated coupling reaction, e.g.Negishi coupling, to provide the tricyclic ring system. The alkylzincreagents are prepared from zinc insertion into the carbon-halide bond ofan ester-substituted alkyl halide, which are either commerciallyavailable or prepared as described in “Synthesis of IodideIntermediates”. The coupling reaction can give a mixture ofdiastereoisomers, which in some instances can be separated directly andin other instances are separated at a further stage. The carboxylicester can then be hydrolyzed under acidic or basic conditions to providea carboxylic acid intermediate. The carboxylic acid intermediate canthen be coupled to a variety of amines, such as those described belowunder the heading “Synthesis of Amine Intermediates,” in Example 8 toprovide the amide final product.

Example 1. General Synthesis of Compound 109 and Related Analogs Step 1:Synthesis of methyl3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate

A solution of 2,6-dibromo-4-methylpyridine (1.50 g, 5.97 mmol) in DCM(30 mL) was cooled to −78° C., and n-BuLi (2.5 M, 2.60 mL, 6.56 mmol)was added dropwise to the above solution at −78° C. The solution wasstirred at −78° C. for another 15 min. Methyl3-oxocyclobutanecarboxylate (917 mg, 7.16 mmol) was added to thesolution, and the resultant mixture was stirred at −78° C. for 30 min.The mixture was then quenched by addition of saturated aqueous NH₄Clsolution and extracted with DCM. Organic layers were combined, driedover sodium sulfate, filtered, and concentrated. The residue waspurified by silica gel column (PE:EA=2:1) to give methyl3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (1.0 g,56%) as a white solid, MS (ES+) C₁₂H₁₄BrNO₃ requires: 299, found: 300[M+H]⁺.

Step 2: Synthesis of3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate

A mixture of methyl3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (400mg, 1.33 mmol), 5-methyl-1H-pyrazol-3-amine (258 mg, 2.66 mmol),t-BuXPhos (69 mg, 0.40 mmol), Pd₂(dba)₃ (182 mg, 0.20 mmol) and KOAc(391 mg, 3.99 mmol) in DMA (6 mL) was heated to 140° C. for 1 h undermicrowave irradiation. After cooling to ambient temperature, the mixturewas concentrated and purified by silica gel column (PE:EA=1:2) to give3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate(120 mg, 29%) as a white solid. MS (ES+) C₁₆H₂₀N₄O₃ requires: 316,found: 317 [M+H]⁺.

Step 3: Synthesis of3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate

To a solution of3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate(120 mg, 0.38 mmol) in MeOH (5 mL) was added 2 M aqueous LiOH (0.5 mL, 1mmol) at 25° C. The solution was stirred at 25° C. for 15 h. Thesolution was concentrated to remove MeOH. The water solution wasacidified by 2 M HCl to bring pH to 6. The precipitated solid wascollected and dried to give3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate(100 mg, 88%) as a yellow solid. MS (ES+) C₁₅H₁₈N₄O₃ requires: 302,found: 303 [M+H]⁺.

Step 4: Synthesis of(1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-4-hydroxy-4-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclohexanecarboxamide(Compound 109)

A mixture of3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate(100 mg, 0.33 mmol),(S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethanamine hydrochloride(80 mg, 0.33 mmol), HATU (125 mg, 0.33 mmol) and DIEA (128 mg, 0.99mmol) in DMA (3 mL) was stirred 25° C. for 2 h. The solution wasconcentrated and purified by preparative HPLC to give the title product(60 mg, 37%) as a white solid. MS (ES+) C₂₅H₂₇FN₈O₂ requires: 490,found: 491 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.70 (s, 1H), 8.86(s, 1H), 8.68 (d, 1H, J=4.8 Hz), 8.39 (d, 1H, J=0.8 Hz), 8.35 (d, 1H,J=8.0 Hz), 7.95-7.86 (m, 3H), 6.94 (s, 1H), 6.74 (s, 1H), 6.14 (s, 1H),5.68 (s, 1H), 5.03-5.00 (m, 1H), 3.00-2.98 (m, 1H), 2.63-2.55 (m, 2H),2.42-2.32 (m, 2H), 2.21 (s, 3H), 2.18 (s, 3H), 1.39 (d, 3H, J=7.2 Hz).

Example 2. General Synthesis of Compound 113 and Related Analogs Step 1:Synthesis of(1S,3R)-3-fluoro-N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxamide

A mixture of(1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-hydroxy-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)cyclobutanecarboxamide(40 mg, 0.082 mmol) in DCM (4 mL) was cooled to 0° C. DAST (39 mg, 0.24mmol) was added to the mixture at 0° C. The resultant mixture wasstirred at 25° C. for 1 h. The mixture was concentrated and purified byPrep-HPLC to give the title compound (15.6 mg, 39%) as a white solid. MS(ES+) C₂₅H₂₆F₂N₈O requires: 492, found: 493 [M+H]⁺. ¹H-NMR (400 MHz,DMSO-d₆) δ ppm 12.50-11.50 (br, 1H), 10.00-9.00 (br, 1H), 8.66 (d, 1H,J=4.0 Hz), 8.56 (d, 1H, J=6.0 Hz), 8.39 (d, 1H, J=2.0 Hz), 7.94-7.85 (m,3H), 6.82 (s, 1H), 6.69 (s, 1H), 6.34 (s, 1H), 5.09-5.05 (m, 1H),3.46-3.40 (m, 2H), 3.29-2.95 (m, 2H), 2.55-2.50 (m, 1H), 2.24 (s, 3H),2.16 (s, 3H), 1.42 (d, 3H, J=7.2 Hz).

Example 3. Synthesis of Compound 117 and Related Analogs Step 1:Synthesis of methyl3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylate

A mixture of methyl3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (2.2 g,7.3 mmol) in DMF (66 mL) was cooled to 0° C. Sodium hydride (60%dispersion in oil, 1.45 g, 36.6 mmol) min was added, and after 15 miniodomethane (2.08 g, 14.7 mmol) was added. The cooling bath was removedand the reaction mixture was stirred for 3 h at ambient temperature. Themixture was then partitioned between ethyl acetate and water, and theorganic layer was washed with brine. The washed organic layer was driedover sodium sulfate, filtered, and the filtrate was concentrated. Theresidue was purified by flash-column chromatography on silica gel(gradient elution, 0 to 100% ethyl acetate-hexanes) to give methyl3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylate (1.6g, 70%).

Steps 2-4: Synthesis of(1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-methoxy-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)cyclobutane-1-carboxamide

The title compound was prepared from methyl3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylateusing the procedures described in Synthetic Protocol 1 and Example 1. MS(ES+) C₂₆H₂₉FN₈O₂ requires: 504, found: 505 [M+H]⁺. ¹H-NMR (400 MHz,DMSO-d₆) δ 8.68 (d, J=4.5 Hz, 1H), 8.51 (d, J=7.7 Hz, 1H), 8.40 (d,J=1.7 Hz, 1H), 7.95-7.88 (m, 3H), 7.25 (s, 1H), 7.07 (s, 1H), 5.98 (s,1H), 5.03-4.98 (m, 1H), 3.07 (s, 3H), 2.97-2.83 (m, 2H), 2.58 (t, J=9.2Hz, 3H), 2.49 (s, 3H), 2.27 (s, 3H), 1.41 (d, J=7.0 Hz, 3H).

Example 4. General Synthesis of Compound 122 and Related Analogs Step 1:Synthesis of ethyl(1s,3s)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate

Ethyl 3-iodo-1-methoxycyclobutane-1-carboxylate (1.00 g, 3.52 mmol) wasdissolved in dimethylacetamide (11.7 mL) in a pressure vessel under astream of nitrogen. Rieke Zinc (5.1 mL of a 50 mg/mL suspension in THF,3.87 mmol) was added quickly via syringe. The vessel was capped andstirred at ambient temperature for 15 minutes. The vessel was openedunder a stream of nitrogen and 2,4-dichloro-6-methylpyrimidine (689 mg,4.22 mmol) was added followed by PdCl₂dppf (258 mg, 0.352 mmol). Thevessel was capped and heated to 70° C. for 3 h. LCMS of the crudereaction mixture showed a ˜3:1 ratio of product isomers. The reactionmixture was then cooled to room temperature, diluted with ethyl acetate,and filtered through celite. The filtrate was transferred to aseparatory funnel and washed with water (3×), brine, and dried oversodium sulfate. The dried solution was filtered, and the filtrate wasconcentrated. The residue was purified by flash-column chromatography onsilica gel (gradient elution, 0 to 50% ethyl acetate-hexanes) to giveethyl(1S,3S)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate(323 mg, 32%) as a colorless oil. The minor isomer was discarded. MS(ES+) C₁₅H₂₂N₂O₃S requires: 310, found: 311 [M+H]⁺.

Steps 2-4: Synthesis of(1R,3S)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-(6-methyl-2-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-4-yl)cyclobutane-1-carboxamide

The title compound was prepared from ethyl(1S,3S)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate(192 mg, 0.53 mmol) using the same three-step procedure (cross-coupling,hydrolysis, and amide coupling) as in Synthetic Protocol 1 and Example 1to give a white solid. MS (ES+) C₂₅H₂₈FN₉O₂ requires: 505, found: 506[M+H]⁺. ¹H-NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 (s, 1H), 8.67(dd, J=4.5, 0.8 Hz, 1H), 8.52 (d, J=8.2 Hz, 1H), 8.44 (d, J=2.2 Hz, 1H),7.99 (dd, J=8.6, 2.3 Hz, 1H), 7.94-7.74 (m, 2H), 6.54 (s, 2H), 5.17-5.01(m, 1H), 3.16-3.03 (m, 4H), 2.71-2.60 (m, 1H), 2.57-2.36 (m, 4H), 2.26(s, 3H), 2.17 (s, 3H), 1.47 (d, J=7.1 Hz, 3H).

Example 5. General Synthesis of Compound 108 and Related Analogs Step 1:Synthesis of2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine

A suspension of 2,4-dichloro-6-methyl-pyrimidine (120.00 g, 736.2 mmol,1.00 eq), 5-methyl-1H-pyrazol-3-amine (78.65 g, 0.81 mol, 1.10 eq) andDIPEA (142.72 g, 1.10 mol, 1.50 eq) in DMSO (400 mL) was heated at 60°C. for 16 hrs. TLC (PE/EA, 5:1, 1:1) showed the reaction was complete.The reaction mixture was cooled to 30° C. and poured into ice-water (800mL). The resulting mixture was extracted with MTBE (800 mL×10). Thecombined organic layers were washed with water (400 mL×3), brine (400mL×3) and dried over Na₂SO₄. After filtration, the filtrate wasconcentrated under reduced pressure and the residue was recrystallizedfrom DCM (10 mL/g) to afford2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine (105.60g, 472.14 mmol, 64%) as a yellow solid. The structure was confirmed byLC-MS and NMR.

Step 2: Synthesis of methyl1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate

Methyl 3-iodo-1-methylcyclobutane-1-carboxylate (659 mg, 2.59 mmol) wasdissolved in dimethylacetamide (3.2 mL) in a microwave reaction vial.The vial was evacuated and backfilled with nitrogen. Rieke Zinc (4.2 mLof a 50 mg/mL suspension in THF, 3.2 mmol) was added quickly viasyringe. The vessel was capped and stirred at ambient temperature for 15minutes. The vessel was opened under a stream of nitrogen and2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine (290 mg,1.30 mmol) was added followed by PdCl₂dppf (285 mg, 0.389 mmol). Thevessel was capped and heated to 80° C. for 2 h. The reaction mixture wasthen cooled to room temperature, diluted with ethyl acetate, andfiltered through celite. The filtrate was transferred to a separatoryfunnel and washed with water (3×), brine, and dried over sodium sulfate.The dried solution was filtered, and the filtrate was concentrated. Theresidue was purified by flash-column chromatography on silica gel(gradient elution, 0 to 10% methanol-ethyl acetate) to give methyl1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate(80 mg, 20%) as a light brown solid. The mixture of diastereomers wasnot separated at this point. MS (ES+) C₁₆H₂₁N₅O₂ requires: 315, found:316 [M+H]⁺.

Steps 3 and 4: Synthesis of(1R,3S)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxamide(Compound 108) and(1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxamide(Compound 107)

The title compounds were prepared from methyl1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate(82 mg, 0.26 mmol) using the same two-step procedure (hydrolysis andamide coupling) as in Synthetic Protocol 1. The diastereomers wereseparated by reverse-phase HPLC (gradient elution, 10% to 50%acetonitrile-water with 0.1% TFA additive). The fractions containing thecompounds were combined and partitioned between ethyl acetate andsaturated aqueous sodium bicarbonate solution. The organic layers werewashed with brine, dried over sodium sulfate, filtered, andconcentrated. Peak 1 was Compound 107 (white solid, 5 mg, 4% over twosteps). Peak 2 was Compound 108 (white solid, 23 mg, 18% over twosteps). The spectral data for these two compounds is presented in thecompound table.

Example 6. Synthesis of Ketone Intermediates Ethyl1-methoxy-3-oxocyclobutane-1-carboxylate

Step 1: Synthesis of 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylicacid

A solution of KOH (12.74 g, 227.0 mmol) in MeOH (70 mL) was added tomixture of 3-(benzyloxy)cyclobutan-1-one (5.00 g, 28.4 mmol) andbromoform (57.4 g, 227 mmol) was added at 0° C. The cooling bath wasremoved and the mixture was stirred at 20° C. for 16 h. LCMS showedreaction was completed and major was desired product. Water (100 mL) wasthen added and the mixture was and extracted with DCM (3×80 mL). Theaqueous phase was adjusted pH to 3 with aqueous hydrochloric acidsolution (0.5 N), then extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with brine (3×50 mL), dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Crudeproduct (4.8 g) was obtained as brown solid which was used to next stepdirectly without further purification

Step 2: Synthesis of ethyl3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate

Potassium carbonate (5.62 g, 40.6 mmol) and iodoethane (4.75 g, 30.5mmol) were added to a solution of3-(benzyloxy)-1-methoxycyclobutane-1-carboxylic acid (4.80 g, 20.3 mmol)in DMF (40 mL) at 0° C. The reaction mixture was stirred at 20° C. for16 h, then was partitioned between water and ethyl acetate. The aqueouslayer was further extracted with ethyl acetate (2×), and the organiclayers were combined. LCMS showed reaction was completed. The mixturewas added H₂O (100 mL) and extracted with EA (100 mL*3). The combinedorganic layers were washed with brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by flash column chromatography on silica gel (PE:EA=10:1) togive ethyl 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate (4.80 g, 79%yield) as a yellow oil.

Step 3: Synthesis of ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate

Palladium hydroxide on carbon (200 mg) was added to a solution of ethyl3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate (2.0 g, 7.6 mmol) inEtOH (80 mL) at ambient temperature in a high pressure reaction vessel.The vessel was evacuated and backfilled with hydrogen for three cycles,and was then charged with hydrogen at 50 psi. The reaction mixture wasstirred for 16 h at 50 psi, then was evacuated and backfilled withnitrogen. The reaction mixture was filtered, and the filtrate wasconcentrated to give ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate(1.0 g, 76% yield) as a light yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ4.46-4.42 (m, 1H), 4.27 (q, J=7.2 Hz, 2H), 3.27 (s, 3H), 2.66-2.58 (m,2H), 2.41-2.34 (m, 2H), 1.33 (t, J=7.2 Hz, 3H).

Step 4: Synthesis of ethyl 1-methoxy-3-oxocyclobutane-1-carboxylate

DMP (6.04 g, 14.2 mmol) was added to a solution of ethyl3-hydroxy-1-methoxycyclobutane-1-carboxylate (1.60 g, 9.19 mmol) in DCM(10 mL) at 20° C. The reaction mixture was stirred for 16 h, and TLCanalysis showed formation of a new spot (PE:EA=10:1, Rf=0.35, stained byI₂). The reaction mixture was filtered and the organic phase wasconcentrated to give crude under reduced pressure. The residue waspurified by flash column chromatography on silica gel (PE:EA=10:1) togive the title compound (1.40 g, 75.2% yield) as a yellow oil.

Example 7. Synthesis of Iodide Intermediates Ethyl3-iodo-1-methoxycyclobutane-1-carboxylate

Step 1: Synthesis of ethyl1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylate

A solution of ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate (29.4g, 129 mmol) and 2,6-lutidine (29.5 mL, 253 mmol) in DCM (500 mL) wascooled to −10° C. Trifluoromethanesulfonic anhydride (50 g, 177 mmol)was then added dropwise over 45 min to the cooled solution. The reactionmixture was stirred for 1 h, then was quenched with saturated ammoniumchloride solution (500 mL) and the layers were partitioned. The organiclayer was dried over sodium sulfate, filtered, and the filtrate wasconcentrated to give ethyl1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylate asa brown oil that was used directly in the next step without anypurification.

Step 2: Synthesis of ethyl 3-iodo-1-methoxycyclobutane-1-carboxylate

A solution of the entire crude ethyl1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylatefrom above (assumed to be 169 mmol) in DMF (170 mL) put in a water bathat ambient temperature. Sodium iodide (76 g, 507 mmol) was added to thesolution and the reaction mixture was stirred at ambient temperature for16 h. The reaction mixture was diluted with water (1 L) and extractedwith ethyl acetate (2×400 mL). The organic layers were combined andwashed sequentially with water (3×1 L), saturated sodium thiosulfatesolution, and brine. The organic layer was then dried over sodiumsulfate and filtered, and the filtrate was concentrated. The residue waspurified by flash-column chromatography on silica gel (gradient elution,0 to 20% ethyl acetate-hexanes) to give the title compound (31.5 g, 66%yield over two steps) as a colorless oil. MS (ES+) C₈H₁₃IO₃ requires:284, found: 285 [M+H]⁺. ¹H NMR (400 MHz, Chloroform-d) δ 4.33 (p, J=8.4Hz, 1H), 4.25 (q, J=7.1 Hz, 2H), 3.24 (s, 3H), 3.20-3.13 (m, 2H),2.82-2.71 (m, 2H), 1.31 (t, J=7.1 Hz, 3H).

Methyl 3-iodo-1-methylcyclobutane-1-carboxylate

Step 1: Synthesis of methyl1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate

Methanesulfonyl chloride (0.60 mL, 7.6 mmol) was added to a solution ofmethyl 3-hydroxy-1-methylcyclobutane-1-carboxylate (1.00 g, 6.94 mmol)and triethylamine (1.26 mL, 9.02 mmol) at 0° C. After the addition, thecooling bath was removed and the reaction mixture was stirred for 70min. The reaction mixture was then partitioned between DCM and aqueoushydrochloric acid solution (1 N). The organic layer was washed withsaturated aqueous sodium bicarbonate solution, brine, and then was driedover sodium sulfate. The mixture was then filtered, and the filtrate wasconcentrated to give methyl1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (1.48 g, 96%)as a light yellow oil that was used in the next step without any furtherpurification.

Step 2: Synthesis of methyl 3-iodo-1-methylcyclobutane-1-carboxylate

Sodium iodide (3.79 g, 25.3 mmol) was added to a solution of methyl1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (1.41 g, 6.32mmol) in DMF (12.6 mL). The reaction mixture was heated to 80° C. for 46h, then was cooled to ambient temperature and partitioned between DCMand water. The organic layer was dried over sodium sulfate, filtered,and concentrated. The residue was purified by flash-columnchromatography on silica get (gradient elution, 0 to 15% ethylacetate-hexanes) to give the title compound (659 mg, 41%) as a colorlessoil. MS (ES+) C₇H₁₁IO₂ requires: 254, found: 255 [M+H]⁺.

Example 8. Synthesis of Amine Intermediates(S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine

Step 1: 1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one

4-Fluoro-1H-pyrazole (4.73 g, 55 mmol) and potassium carbonate (17.27 g,125 mmol) were combined and stirred in N,N-dimethylformamide (41.7 mL)for 10 minutes in an open sealed tube before addition of2-bromo-5-acetylpyridine (10 g, 50 mmol). The reaction tube was sealedand stirred 20 hours at 100° C. The reaction mixture was then cooled toroom temperature and poured into water (˜700 mL). The mixture wassonicated and stirred for 20 minutes. A beige solid was isolated byfiltration, washed with small amounts of water, and dried to yield1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one (9.81 g, 96%yield). MS: M+1=206.0.

Step 2:(R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide

To a stirred room temperature solution of1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one (9.806 g, 47.8mmol) in THF (96 mL) was added (R)-(−)-t-Butylsulfinamide (5.79 g, 47.8mmol) followed by titanium (IV) ethoxide (21.8 g, 96 mmol). The solutionwas stirred at 75° C. on an oil bath for 15 hours. The reaction solutionwas cooled to room temperature and then to −78° C. (externaltemperature) before the next step. To the −78° C. solution was addeddropwise over nearly 55 minutes L-Selectride (143 mL of 1N in THF, 143mmol). During addition, some bubbling was observed. The reaction wasthen stirred after the addition was completed for 15 minutes at −78° C.before warming to room temperature. LC-MS of sample taken during removalfrom cold bath showed reaction was completed. The reaction was cooled to−50° C. and quenched slowly with methanol (˜10 mL), then poured intowater (600 mL) and stirred. An off-white precipitate was removed byfiltration, with ethyl acetate used for washes. The filtrate was dilutedwith ethyl acetate (800 mL), the layers were separated, and the organiclayer was dried over sodium sulfate, filtered, and concentrated down.The crude was purified by silica gel chromatography to yield(R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide(10.5 g, 99% purity, 70.3% yield) as a light yellow solid. MS:M+1=311.1.

Step 3: (S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine

A solution of(R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide(10.53 g, 33.9 mmol)) in methanol (79 mmol) and 4N HCl/dioxane (85 mL,339 mmol) was stirred 2.5 hours. LC-MS showed reaction was completed.The reaction solution was poured into diethyl ether (300 mL). A stickysolid was formed. The mixture was treated with ethyl acetate (200 mL)and sonicated. The solvents were decanted, and the sticky solid wastreated with more ethyl acetate (˜200 mL), sonicated and stirred. Thebulk of the sticky solid was converted to a suspension. A light yellowsolid was isolated by filtration, washed with smaller amounts of ethylacetate, and dried to yield(S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine (7.419 g,78% yield). LC-MS confirmed desired product in high purity. MS:M+1=207.1.

(S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine

Step 1: 1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one

Sodium hydride (60 wt %, 276 mg, 6.90 mmol) was added to a mixture of1-(5-chloropyrazin-2-yl)ethanone (800 mg, 5.11 mmol) and4-Fluoro-1H-pyrazole (484 mg, 5.62 mmol) in N,N-dimethylformamide (6.0mL) at ambient temperature for 10 minutes. The reaction mixture was thenpoured into water (70 mL) and was sonicated and stirred for 20 minutes.A dark red solid was isolated by filtration, washed with small amountsof water, and dried to1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one (919 mg, 95%yield). MS: M+1=207.

Step 2:(R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide

To a stirred room temperature solution of1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one (4.67 g, 22.7mmol) in THF (45 mL) was added (R)-(−)-t-Butylsulfinamide (2.75 g, 22.7mmol) followed by titanium (IV) ethoxide (10.3 g, 45.3 mmol). Thesolution was stirred at 75° C. on an oil bath for 20 hours. The reactionsolution was cooled to room temperature and then to −78° C. before thenext step. To the −78° C. solution was added dropwise over 50 minutesL-Selectride (50.1 mL of 1 N in THF, 50.1 mmol). During addition, somebubbling was observed. The reaction was then stirred after the additionwas completed for 15 minutes at −78° C. before warming to roomtemperature. LC-MS of sample taken during removal from cold bath showedreaction was completed. The reaction was cooled to −60° C. and quenchedslowly with methanol (1 mL), then poured into water (100 mL) andstirred. The mixture was filtered and the solids were washed furtherwith ethyl acetate. The filtrate was diluted with ethyl acetate, thelayers were separated, and the organic layer was dried over sodiumsulfate. The dried solution was filtered, and the filtrate wasconcentrated. The residue was purified by flash-column chromatography(gradient elution, 0 to 100% ethyl acetate-dichloromethane) to give(R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide(1.04 g, 14%) as a brown solid. MS: M+1=312. ¹H NMR (400 MHz, DMSO-d6) δ9.12 (d, J=1.4 Hz, 1H), 8.73 (d, J=4.5 Hz, 1H), 8.59 (d, J=1.4 Hz, 1H),8.03 (d, J=4.1 Hz, 1H), 5.69 (d, J=5.7 Hz, 1H), 4.62 (p, J=6.8 Hz, 3H),1.57 (d, J=6.9 Hz, 3H), 1.12 (s, 9H).

Step 3: (S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine

A solution of(R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide(1.04 g, 3.34 mmol) in methanol (7.8 mL) and 4N HCl/dioxane (8.34 mL,33.4 mmol) was stirred for 1.5 h at ambient temperature. The reactionmixture was poured into diethyl ether (100 mL). A light beige solid wasisolated by filtration to afford(S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine (689 mg,85% yield). MS: M+1=208.

(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine

Step 1: 5-(4-fluoro-1H-pyrazol-1-yl)pyrazine-2-carbonitrile

To a solution of 5-chloropyrazine-2-carbonitrile (280 mg, 2.0 mmol) inDMF was added 4-fluoro-1H-pyrazole (170 mg, 2.0 mmol), and potassiumacetate (395 mg, 4.0 mmol). The mixture was stirred at the 100° C. for 4hours. The reaction mixture was cooled to 20° C., poured into brine (25mL), and extracted with ethyl acetate. The organic layer was dried oversodium sulfate, concentrated and purified by column chromatography(hexane:ethyl acetate=5:1) to give5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-carbonitrile (310 mg, Yield 82%).The structure was confirmed by LC-MS.

Step 2: (5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine

A mixture of 5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-carbonitrile (190 mg,1.0 mmol) and NiCl₂ (12 mg, 0.1 mmol) in MeOH (5 mL) was added NaBH₄(380 mg, 10 mmol) at 0° C. The mixture was stirred at 0° C. for 2 hours,quenched with aqueous NH₄Cl and purified by HPLC to give(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine (160 mg, Yield82%). The structure was confirmed by LC-MS.

(6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

Step 1: 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile

To the solution of 6-chloronicotinonitrile (300 mg, 2.2 mmol) in DMF (10mL), was added 3,5-dimethyl-1H-pyrazole (210 mg, 2.2 mmol) and Cs₂CO₃(1.4 g, 4.4 mmol). The mixture was stirred at 90° C. for 16 h. Water (25mL) was added to the reaction mixture, and the mixture was filtered. Thesolids were washed with water and dried under vacuum to give6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile (320 mg, yield 74.6%).

Step 2: tert-Butyl((6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate

To 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile (300 mg, 1.5 mmol) inMeOH (10 mL), was added NiCl₂ (19 mg, 0.15 mmol), (Boc)₂O (654 mg, 3.0mmol) and NaBH₄ (142 mg, 3.8 mmol). The mixture was stirred at ambienttemperature for 3 h. Saturated aqueous ammonium chloride solution wasadded and the MeOH was removed under vacuum. The aqueous suspension wasthen partitioned with ethyl acetate, and the layers were separated. Theorganic layer was washed with saturated sodium bicarbonate solution(2×50 mL). The organic layer was dried with anhydrous sodium sulfate,filtered, and concentrated under vacuum to give 450 mg target compoundwhich was used in the next step without further purification.

Step 3: 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile

A solution of HCl in Dioxane (4.0 M, 10 mL) was added to compoundtert-Butyl((6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate (450mg). The mixture was stirred for 2 h, then was dried under reducedpressure to give the title compound (350 mg) as a light brown solid thatwas used without further purification. ¹H NMR (400 MHz, DMSO-d6) δ 8.51(d, J=2.1 Hz, 1H), 8.34 (s, 3H), 8.03 (dd, J=8.5, 2.4 Hz, 1H), 7.87 (d,J=8.5 Hz, 1H), 6.14 (s, 1H), 4.12 (q, J=5.7 Hz, 2H), 2.59 (s, 3H), 2.21(s, 3H).

(6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

Step 1: 6-(4-Chloro-1H-pyrazol-1-yl)nicotinonitrile

To a solution of 6-chloronicotinonitrile (300 mg, 2.2 mmol) in DMF (10mL), was added 4-chloro-1H-pyrazole (227 mg, 2.2 mmol) and Cs₂CO₃ (1.4g, 4.4 mmol). The mixture was stirred at 90° C. for 16 h. Water (25 mL)was added to the mixture, and the mixture was filtered. The solids werewashed with water and dried under vacuum to give6-(4-Chloro-1H-pyrazol-1-yl)nicotinonitrile (380 mg, 84%).

Step 2: tert-Butyl((6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate

To 6-(4-chloro-1H-pyrazol-1-yl)nicotinonitrile (350 mg, 1.7 mmol) inMeOH (10 mL), was added NiCl₂ (19 mg, 0.17 mmol), (Boc)₂O (741 mg, 3.4mmol) and NaBH₄ (163 mg, 4.3 mmol). The mixture was stirred at ambienttemperature. for 3 h. Saturated aqueous ammonium chloride solution wasadded and the MeOH was removed under vacuum. The aqueous suspension wasthen partitioned with ethyl acetate, and the layers were separated. Theorganic layer was washed with saturated sodium bicarbonate solution(2×50 mL). The organic layer was dried with anhydrous sodium sulfate,filtered, and concentrated under vacuum to give 480 mg target compound,which was used in the next step without further purification.

Step 3: (6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

A solution of HCl in Dioxane (4.0 M, 10 mL) was added to tert-Butyl((6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate (450 mg, 1.5mmol) at ambient temperature. The mixture was stirred for 2 h, then wasdried under reduced pressure to give the title compound (290 mg) as alight brown solid that was used without further purification. MS:M+1=209.

The synthetic protocols that were used to prepare the compoundsdisclosed herein are indicated below. The NMR and LC MS data obtainedfor compounds disclosed herein are also shown below.

TABLE 5 Compound Synthetic MS Number Protocol ¹H NMR (M + 1) 100 2 ¹HNMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.85 433 (s, 1H), 8.59 (d, J =2.6 Hz, 1H), 8.42-8.32 (m, 2H), 7.94-7.83 (m, 2H), 7.80 (d, J = 1.6 Hz,1H), 6.90 (s, 1H), 6.56 (dd, J = 2.6, 1.7 Hz, 1H), 6.40 (s, 1H), 6.15(s, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.57-3.43 (m, 1H), 3.15- 3.07 (m, ,1H), 2.44 (d, J = 8.5 Hz, 4H), 2.17 (d, J = 2.6 Hz, 6H 101 2 ¹H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 8.85 457 (s, 1H), 8.59 (d, J = 2.6 Hz,1H), 8.40 (d, J = 1.9 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.96-7.84 (m,2H), 7.80 (d, J = 1.6 Hz, 1H), 6.90 (s, 1H), 6.55 (t, J = 2.1 Hz, 1H),6.38 (s, 1H), 6.16 (s, 1H), 5.09-4.97 (m, 1H), 3.52- 3.39 (m, 1H),3.17-3.07 (m, 1H), 2.46-2.32 (m, 4H), 2.18 (s, 3H), 2.16 (s, 3H), 1.41(d, J = 7.0 Hz, 3H). 102 2 ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.66 (s,1H), 475 8.87 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.40 (d, J = 2.2 Hz,1H), 8.32 (d, J = 7.7 Hz, 1H), 8.00-7.79 (m, 3H), 6.90 (s, 1H), 6.38 (s,1H), 6.16 (s, 1H), 5.07-4.98 (m, 1H), 3.55-3.40 (m, 1H), 3.16-3.06 (m,1H), 2.46- 2.31 (m, 4H), 2.17 (s, 3H), 2.16 (s, 3H), 1.40 (d, J = 7.0Hz, 3H). 103 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.60 (s, 1H), 475 8.85(s, 1H), 8.65 (d, J = 4.5 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.27 (d, J= 7.7 Hz, 1H), 7.95-7.79 (m, 3H), 6.82 (s, 1H), 6.36 (s, 1H), 6.25 (s,1H), 5.05-4.95 (m, 1H), 3.06-2.95 (m, 1H), 2.47-2.21 (m, 4H), 2.15 (s,3H), 2.12 (s, 3H), 1.40 (d, J = 7.1 Hz, 3H). 104 2 ¹H NMR (400 MHz,DMSO-d6) δ 11.65 (s, 1H), 9.13 476 (d, J = 1.4 Hz, 1H), 8.85 (s, 1H),8.72 (dd, J = 4.6, 0.8 Hz, 1H), 8.48 (d, J = 1.4 Hz, 1H), 8.36 (d, J =7.3 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.90 (s, 1H), 6.38 (s, 1H), 6.16(s, 1H), 513-5.06 (m, 1H), 3.46 (t, J = 7.9 Hz, 1H), 3.18-3.11(m, 1H),3.00 (td, J = 6.6, 3.9 Hz, 2H), 2.18 (s, 2H), 2.16 (s, 3H), 1.77-1.67(m, 2H), 1.44 (d, J = 7.1 Hz, 3H). 105 3 ¹H NMR (400 MHz, DMSO-d6) δ ppm11.90 (s, 1H), 476 9.53 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.39 (d, J =2.1 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 7.96-7.81 (m, 3H), 6.87 (s, 1H),6.13 (s, 1H), 5.06-4.97 (m, 1H), 3.50- 3.40 (m, 1H), 3.19-3.09 (m, 1H),2.49-2.31 (m, 4H), 2.25 (s, 3H), 2.19 (s, 3H), 1.40 (d, J = 7.0 Hz, 3H).107 3 ¹H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 9.56 490 (s, 1H), 8.64(d, J = 4.5 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H),7.99-7.79 (m, 3H), 6.80 (s, 1H), 6.19 (s, 1H), 5.05-4.97 (m, 1H), 3.47(p, J = 8.9 Hz, 1H), 2.60 (q, J = 10.1 Hz, 2H), 2.22 (s, 3H), 2.18- 2.02(m, 5H), 1.53-1.37 (m, 6H). 108 3 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.92(s, 1H), 490 9.56 (s, 1H), 8.69 (d, J = 4.5 Hz, 1H), 8.41 (d, J = 2.2Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.00-7.83 (m, 3H), 6.80 (s, 1H), 6.22(s, 1H), 5.13-4.98 (m, 1H), 3.27- 3.17 (m, 1H), 2.76-2.59 (m, 2H),2.30-2.12 (m, 8H), 1.45 (d, J = 7.1 Hz, 3H), 1.34 (s, 3H). 110 3 ¹H NMR(400 MHz, DMSO-d6) δ ppm 11.91 (s, 1H), 491 9.55 (s, 1H), 9.13 (d, J =1.4 Hz, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.47 (d, J = 1.5 Hz, 1H), 8.24(d, J = 7.4 Hz, 1H), 8.02 (d, J = 4.2 Hz, 1H), 6.80 (s, 1H), 6.21 (s,1H), 5.19-5.03 (m, 1H), 3.28-3.18 (m, 1H), 2.78-2.66 (m, 2H), 2.28-2.15(m, 8H), 1.49 (d, J = 7.1 Hz, 3H), 1.36 (s, 3H). 114 1, ¹H NMR (400 MHz,DMSO-d6) δ 11.73 (s, 1H), 9.05 493 minor (s, 1H), 8.67 (d, J = 4.5 Hz,1H), 8.49 (d, J = 7.7 Hz, isomer 1H), 8.39 (d, J = 2.1 Hz, 1H),7.99-7.80 (m, 3H), 7.01 (s, 1H), 6.67 (s, 1H), 6.14 (s, 1H), 5.09-4.93(m, 1H), 2.92 (p, J = 9.1, 8.6 Hz, 1H), 2.83-2.69 (m, 2H), 2.69- 2.50(m, 2H), 2.20 (d, J = 17.2 Hz, 6H), 1.39 (d, J = 6.9 Hz, 3H). 116 1 ¹HNMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.88 505 (s, 1H), 8.67 (d, J =4.5 Hz, 1H), 8.43-8.25 (m, 1H), 7.98-7.75 (m, 3H), 6.80 (s, 1H), 6.76(s, 1H), 6.09 (s, 1H), 5.92-5.82 (m, 1H), 5.68 (s, 1H), 3.38-3.26 (m,1H), 2.70-2.55 (m, 5 H), 2.25-2.05 (m, 8H), (s, 2H), 2.20 (s, 2H), 2.11(s, 2H), 1.50 (d, J = 7.1 Hz, 3H). 118 1 ¹H NMR (400 MHz, DMSO-d6) δ12.83 (s, 1H), 11.60 505 (s, 1H), 8.67 (d, J = 4.6 Hz, 1H), 8.38 (d, J =2.1 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.92 (dd, J = 8.1, 2.9 Hz, 2H),7.86 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H), 6.75 (s, 1H), 5.92 (s, 1H), 3.00(d, J = 13.1 Hz, 1H), 2.89 (d, J = 13.0 Hz, 1H), 2.34 (d, J = 13.4 Hz,2H), 2.28 (s, 3H), 2.25 (s, 3H), 1.58 (s, 3H), 1.43 (d, J = 7.1 Hz, 3H).119 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.68 (s, 1H), 505 8.87 (s, 1H),8.67 (d, J = 4.5 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.18 (d, J = 7.8 Hz,1H), 8.04-7.94 (m, 1H), 7.94-7.80 (m, 2H), 6.79 (s, 1H), 6.70 (s, 1H),6.29 (s, 1H), 5.46 (s, 1H), 5.15-4.97 (m, 1H), 2.55 (s, 4H), 2.25-2.12(m, 6H), 1.45 (d, J = 7.1 Hz, 3H), 1.35 (s, 3H). 120 2 ¹H NMR (400 MHz,DMSO-d6) δ 11.68 (s, 1H), 8.94 505 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H),8.51 (d, J = 8.3 Hz, 1H), 8.45 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.6,2.3 Hz, 1H), 7.94-7.85 (m, 2H), 6.80 (s, 1H), 6.40 (s, 1H), 6.27 (s,1H), 5.21-4.94 (m, 1H), 3.19-3.00 (m, 4H), 2.70-2.35 (m, 4H), 2.17 (d, J= 6.7 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H). 121 3 ¹H NMR (400 MHz, DMSO-d6)δ 11.93 (s, 1H), 9.61 506 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.49 (d, J= 8.2 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.5, 2.3 Hz, 1H),7.93-7.85 (m, 2H), 6.84 (s, 1H), 6.20 (s, 1H), 5.16-5.01 (m, 1H), 3.10(s, 4H), 2.69-2.39 (m, 4H), 2.24 (s, 3H), 2.19 (s, 3H), 1.47 (d, J = 7.1Hz, 3H). 123 3 ¹H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 9.66- 507 9.48(m, 1H), 9.13 (d, J = 1.3 Hz, 1H), 8.73 (d, J = 4.5 Hz, 1H), 8.54 (d, J= 1.3 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.84(s, 1H), 6.21 (s, 1H), 5.29-5.12 (m, 1H), 3.23-3.08 (m, 4H), 2.70-2.43(m, 4H), 2.24 (s, 3H), 2.19 (s, 3H), 1.51 (d, J = 7.1 Hz, 3H). 124 2 ¹HNMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 507 (s, 1H), 9.13 (d, J =1.4 Hz, 1H), 8.73 (d, J = 4.5 Hz, 1H), 8.54 (d, J = 1.4 Hz, 1H), 8.44(d, J = 7.9 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.54 (s, 2H), 5.25-5.06(m, 1H), 3.17 (s, 4H), 2.71-2.37 (m, 4H), 2.26 (s, 3H), 2.19 (s, 3H),1.51 (d, J = 7.0 Hz, 3H). 126 1 ¹H NMR (400 MHz, DMSO-d6) δ 10.04-9.91(m, 507 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.32 (s, 2H), 7.91 (d, J = 4.1Hz, 1H), 7.84 (s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 6.74 (s,1H), 6.14 (s, 1H), 5.09-5.04 (m, 1H), 3.28-3.22 (m, 1H), 3.14-3.08 (m,1H), 2.40-2.36 (m, 1H), 2.25 (s, 3H), 2.21 (s, 3H), 2.04-1.96 (m, 1H),1.61 (s, 3H). 127 1 ¹H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.99 521(s, 1H), 8.72 (d, J = 8.1 Hz, 1H), 8.68 (d, J = 4.5 Hz, 1H), 8.46 (d, J= 2.2 Hz, 1H), 8.02 (dd, J = 8.5, 2.3 Hz, 1H), 7.94-7.85 (m, 2H), 6.73(s, 1H), 6.46 (s, 1H), 5.81 (s, 1H), 5.21-5.08 (m, 1H), 3.16 (s, 3H),2.93 (t, J = 11.9 Hz, 2H), 2.49 (s, 3H), 2.45-2.34 (m, 2H), 2.18 (s,3H), 1.50 (d, J = 7.1 Hz, 3H). 128 1 ¹H NMR (400 MHz, DMSO-d6) δ 11.61(s, 1H), 8.80 521 (s, 1H), 8.63 (d, J = 4.5 Hz, 1H), 8.37 (t, J = 4.1Hz, 2H), 7.94-7.86 (m, 2H), 7.82 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 6.66(s, 1H), 6.22 (s, 1H), 5.50 (s, 1H), 5.06- 4.99 (m, 1H), 3.14-3.03 (m,5H), 2.32-2.25 (m, 2H), 2.14 (s, 3H), 2.13 (s, 3H), 1.42 (d, J = 7.0 Hz,3H). 131 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 493 (s, 1H),9.12 (d, J = 1.4 Hz, 1H), 8.72 (d, J = 4.5 Hz, 1H), 8.65 (t, J = 6.0 Hz,1H), 8.44 (d, J = 1.4 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.55 (s, 2H),4.53 (d, J = 5.9 Hz, 2H), 3.22-3.14 (m, 4H), 2.67-2.42 (m, 4H), 2.27 (s,3H), 2.19 (s, 3H).

Example 9: Measurement of Biochemical Activity of Compounds

In order to assess the activity of chemical compounds against therelevant kinase of interest, the Caliper LifeSciences electrophoreticmobility shift technology platform is used. Fluorescently labeledsubstrate peptide is incubated in the presence of kinase and ATP so thata reflective proportion of the peptide is phosphorylated. At the end ofthe reaction, the mix of phosphorylated (product) and non-phosphorylated(substrate) peptides are passed through the microfluidic system of theCaliper EZ Reader 2, under an applied potential difference. The presenceof the phosphate group on the product peptide provides a difference inmass and charge between those of the substrate peptide, resulting in aseparation of the substrate and product pools in the sample. As thepools pass a LEDS within the instrument, these pools are detected andresolved as separate peaks. The ratio between these peaks thereforereflects the activity of the chemical matter at that concentration inthat well, under those conditions.

RET Wild Type Assay at KM

In each well of a 384-well plate, 7.5 nM-10 nM of wild type RET(ProQinase 1090-0000-1) is incubated in a total of 12.5 μL of buffer(100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl₂, 1 mM DTT) with 1 μMCSKtide (FITC-AHA-KKKKD DIYFFFG-NH2) (SEQ ID NO: 5) and 25 μM ATP at 25°C. for 120 minutes in the presence or absence of a dosed concentrationseries of compound (1% DMSO final concentration). The reaction isstopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5,0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (CaliperLifesciences)). The plate is then read on a Caliper EZReader 2 (protocolsettings: −1.7 psi, upstream voltage −500, downstream voltage −3000,post sample sip 35s). Data is normalized to 0% and 100% inhibitioncontrols and the IC₅₀ calculated using a 4-parameter fit in the CORELIMS.

RET V804L Gatekeeper Mutant Assay at KM

In each well of a 384-well plate, 7.5 nM-10 nM of mutant RET (ProQinase1096-0000-1) is incubated in a total of 12.5 μL of buffer (100 mM HEPESpH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide(FITC-AHA-KKKKDDIYFFFG-NH2) (SEQ ID NO: 5) and 10 μM ATP at 25° C. for120 minutes in the presence or absence of a dosed concentration seriesof compound (1% DMSO final concentration). The reaction is stopped bythe addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)).The plate is then read on a Caliper EZReader 2 (protocol settings: −1.7psi, upstream voltage −500, downstream voltage −3000, post sample sip35s). Data is normalized to 0% and 100% inhibition controls and the IC₅₀calculated using a 4-parameter fit in the CORE LIMS.

In Table 6 below, the following designations are used: ≤1.00 nM=A;1.01-10 nM=B; 10.01-100 nM=C; <100 nM=D; and ND=not determined.

TABLE 6 RET Inhibitory Activity of Exemplary Compounds of the Invention.Wild-type Compound RET V804L Mutant 100 A A 101 A A 102 A A 103 C B 104A A 105 A A 107 B B 108 A A 109 A A 110 A A 113 C C 114 B B 116 B B 117D C 118 D D 119 A A 120 A A 121 A A 122 A A 123 A A 124 A A 126 D D 127A A 128 D D 131 B ND

Example 10: Compounds of Structural Formula (I) are Potent Inhibitors ofWild-Type and Mutant RET

In embodiments, compounds according to structural Formula (I) andstructural Formula (II) are potent and selective inhibitors of oncogenicRET mutant and fusion proteins. Currently, there are no approvedinhibitors that selectively target these disease-driving RETalterations.

1. In Vitro Assays

A compound described herein can be tested in vitro for inhibition ofwild-type RET and various mutant RET kinases, including e.g., RET V804L,RET V804M, and RET M918T kinases, as well as CCDC6-RET and KIF5B-RETfusion kinases. The IC50 can be calculated.

2. Cellular Assays

In cellular systems, the activity of a compound of structural Formula(I) or structural Formula (II) can be measured by inhibition of RETmutant or RET fusion autophosphorylation, RET-dependent signaling, andby inhibition of RET dependent cell proliferation. The compound can beassayed for activity in cancer cell lines endogenously expressingactivated RET fusions or mutants. Exemplary cells that can be used forthese studies include patient xenografts and established cell lines.Exemplary patient xenografts include Colorectal Cancer patient derivedxenograft, Lung adenocarcinoma patient derived xenograft, and NSCLCpatient derived xenograft. Exemplary cell lines include Ba/F3-KIF5B-RET(a model for leukemia), LC2/ad cells (a model for lung cancer), MZ-CRC 1(a model for thyroid cancer), and TT cells (a medullary thyroid cancercell line). Exemplary RET mutations that can be used for these studiesinclude fusions such as KIF5B-RET and CCDC6-RET; point mutations such asRET C634W, RET V804L, RET V804E, RET V804M, and RET M918T; and fusionscontaining point mutations such as KIF5B-RET (V804L) and KIF5B-RET(V804M). KIF5B-RET (V804L) refers to a mutant RET that comprises afusion with KIF5B and further comprises a V804L mutation in RET(referring the amino acid numbering of wild-type RET). KIF5B-RET (V804M)refers to a mutant RET that comprises a fusion with KIF5B and furthercomprises a V804M mutation in RET.

2a. Autophosphorylation Assays

As an example of an autophosphorylation assay, in Ba/F3 modelsengineered to express KIF5B RET, the compound is tested for its abilityto inhibit RET fusion protein signaling as measured by inhibition of RETautophosphorylation. IC50 can be calculated. Wild-type RET inhibitorscabozantinib and vandetanib can be used as controls (less potentcompounds than the test compound) in these cellular assays. Inembodiments, the compound of structural Formula (I) and (II) potentlyand selectively inhibit RET autophosphorylation.

The compound can also be tested for ability to inhibit RETautophosphorylation in LC2/ad cells, a non-engineered NSCLC cell linethat expressed a CCDC6-RET fusion (Suzuki et al, 2013). For instance,immunoblot is performed for LC2/ad cells expressing the CCDC6-RET fusionprotein and treated with the test compound at different concentrations;Phosphorylated (phospho[Y1062]) and total protein levels of RET aremeasured.

2b. Proliferation Assays

In embodiments, the compound of structural Formula (I) and (II) inhibitsproliferation.

In proliferation assays, the compound can be tested for ability toinhibit KIF5B-RET dependent Ba/F3 cell growth. The IC50 can becalculated.

Inhibition of RET activity with the compound can also be tested forinhibition of proliferation of the CCDC6-RET expressing cell line.Similarly, the compound can also be tested for its ability to inhibitRET pathway signaling and RET dependent proliferation in the human MTCTT and MZ-CRC 1 cell lines, driven by RET C634W or RET M918T mutations,respectively. In some embodiments, in RET-driven cell lines, the testcompound inhibits RET activity and RET-driven proliferation morepotently than the multi-kinase inhibitors such as cabozantinib andvandetanib.

2c. Downstream Signalling Assays

In LC2/ad cells, a non-engineered NSCLC cell line that expressed aCCDC6-RET fusion (Suzuki et al, 2013), the compound can be tested forits ability to inhibit phosphorylation of the RET substrate Src homologydomain (Shc) (Hayashi et al, 2000), and downstream signaling throughextracellular signal regulated kinase (ERK) 1/2, includingdownregulation of dual specificity phosphatase 6 (DUSP6) and sproutyreceptor tyrosine kinase signaling antagonist 4 (SPRY4) (Lito et al,2013). For instance, immunoblot can be performed for LC2/ad cellsexpressing the CCDC6-RET fusion protein and treated with the testcompound at different concentrations; phosphorylated and total levels ofdownstream biomarkers, e.g., phospho(Y239/Y240)-Shc andphospho(Y202/T204)-ERK1/2, are measured. In addition, to determineexpression levels of downstream targets, LC2/ad cells can be treatedwith the compound, cabozantinib, or DMSO for 7 hours and RNA isharvested. Gene expression levels of DUSP6 and SPRY4 can be measured byqRT-PCR. In embodiments, the compound induces a dose-dependent decreasein expression of the ERK1/2 target genes DUSP6 and SPRY4 but not thecontrol gene glycogen synthase kinase 3 beta (GSK3B).

3. Animal Models

Antitumor efficacy of compounds of structural Formula (I) and (II) canbe demonstrated in several RET-driven in vivo models. TheBa/F3-KIF5B-RET allograft model uses KIF5B RET fusion-dependent Ba/F3cells. The test compound can be administered orally with an appropriatedose. Tumor size can be measured e.g., twice weekly. In embodiments,administration of the compound results in robust and dose-dependentgrowth inhibition of Ba/F3-KIF5B-RET allograft tumors, e.g., in completeTGI and Mouse body weight can be measured e.g., twice-weekly during theadministration. In embodiments, the compound is well tolerated with nosignificant changes in animal body weight observed.

Similar assays can be performed using other animal models, including aBa/F3-KIF5B-RET (V804L) allograft tumor model which comprises a KIF5BRET V804L fusion protein, an KIF5B-RET NSCLC allograft tumor model, anMTC cell line xenograft driven by a RET C634W mutation, and a CCDC6-RETfusion positive colorectal cancer allograft tumor model. The RET V804Lmutation has been observed in rare cases of MTC and is predicted to beinsensitive to cabozantinib and vandetanib in vitro and in vivo(Carlomagno et al, 2004; Bentzien et al, 2013; BPM 0016). Inembodiments, the compound causes complete TGI and regressions in acancer that is not responsive to cabozantinib or vandetanib.

Biochemical markers can also be assayed in the treated mice. To assessdirect inhibition of KIF5B-RET (V804L) fusion kinase activity in Ba/F3KIF5B-RET (V804L) tumors, the compound can be administered orally at anappropriate dose to tumor bearing mice for several days and plasma andtumors can be collected from individual mice at appropriate time pointsafter the last dose. Test compound concentrations in plasma can bedetermined by liquid chromatograph/tandem mass spectrometry (LC/MS/MS).Inhibition of KIF5B-RET (V804L) signaling in the tumor tissue can beassessed by a phosphor RET enzyme linked immunosorbent assay (ELISA) andby immunoblotting, e.g., as described above. Quantitation of thephospho-RET signal by ELISA can measure the percent KIF5B-RET (V804L)inhibition in treated animals as compared to vehicle treated controls.Suppression of downstream RET pathway signaling can be demonstrated byinhibition of Shc phosphorylation. In embodiments, a dose andtime-dependent correlation is observed between the concentration of thetest compound in mouse plasma and the level of phosphorylated KIF5B RET(V804L). In embodiments, administration of the compound at an amountsufficient to reach at least 90% inhibition of RET in vivo leads totherapeutic efficacy, e.g., can lead to 100% tumor growth inhibition.

Example 11: Selectivity of Compounds of Structural Formulas (I) and (II)

Efficacy Against Wild-Type and Mutant RET

In embodiments, compounds according to structural Formula (I) andstructural Formula (II) are potent inhibitors of wild-type and mutantRET. For instance the IC50 of a compound can be tested in a cell linecomprising wild-type RET and in a second cell line comprising mutantRET, e.g., a point mutation or fusion.

Selectivity for RET Over KDR

In embodiments, compounds according to structural Formula (I) andstructural Formula (II) are selective for RET over another kinase, suchas KDR (also called Vascular endothelial growth factor receptor 2). KDRis a tyrosine-protein kinase that acts as a cell-surface receptor forVEGFA, VEGFC and VEGFD. Inhibition of KDR/VEGFR2 has been associatedclinically with certain adverse effects, e.g., hypertension, arterialthrombosis, and hemorrhage, and therefore selectivity for RET over KDRis desirable.

To test selectivity, the test compound can be assayed for its ability toinhibit proliferation in parental Ba/F3 cells that do not express a RETmutation, e.g., does not express a KIF5B-RET fusion. A weak IC50 in theparental cell line indicates that the test compound is selective forcell lines dependent on oncogenic RET.

The selectivity of a compound on RET versus other human kinases can becharacterized by profiling binding across a panel of over 450 humankinases and disease-relevant kinase mutants. In embodiments, thecompound has a high degree of selectivity for RET and RET kinase mutantsover other kinases tested. To define the binding affinity for thekinases bound by the compound in kinome screening and additional kinasesof interest, the dissociation constant (Kd) can be determined.

To differentiate the compound from multi-kinase inhibitors withbiochemical activity against RET, the activity of the compound againstrecombinant kinase insert domain receptor (KDR) (also known as vascularendothelial growth factor receptor 2 [VEGFR2]) and fibroblast growthfactor receptor 1 (FGFR1) can be tested, as inhibition of these kinasesis associated with dose-limiting toxicities in humans. Inhibition ofKDR/VEGFR2 has been associated clinically with hypertension, arterialthrombosis, and hemorrhage, whereas inhibition of fibroblast growthfactor receptors (FGFRs) is associated with hyperphosphatemia and tissuecalcification (Abdel-Rahman and Fouad, 2014; Touat et al, 2015). Inembodiments, the compound is a more potent inhibitor of WT RET thanKDR/VEGFR2 and FGFR1, respectively. In contrast, in embodiments amulti-kinase inhibitor exhibits approximately equal or increased potencyon KDR versus WT RET.

Example 12: Selective Compounds Prevent RET Resistance Mutants

The compounds herein can be tested for the propensity of a cancer todevelop one or more RET mutations associated with drug resistance. Forexample, RET-altered cancer cells (e.g., Ba/F3 KIF5B-RET cells) can betreated with a mutagen such as ENU, exposed to a compound herein or acontrol compound (e.g., for 2-3 weeks), and the cell number can bequantified. Cells with high proliferation can be subjected to DNAsequencing to detect RET mutations. In embodiments, a compound ofstructural Formula (I) or (II) leads to no or fewer RET mutations than acontrol compound such as a multi-kinase inhibitor such as cabozantinib.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A compound having the structural Formula(I):

or a pharmaceutically acceptable salt thereof, wherein: ring A is anaryl or heteroaryl ring; each of X¹ and X² is independently N or C(R⁶);each R¹ and each R⁷ is independently selected from C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, halo, C₁-C₆ heteroalkyl,cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl,heterocyclylalkyl, nitro, cyano, —C(O)R, —OC(O)R, —C(O)OR, —(C₁-C₆alkylene)-C(O)R, —SR, —S(O)₂R, —(O)₂—N(R)(R), —(C₁-C₆ alkylene)—S(O)₂R,—(C₁-C₆ alkylene)-S(O)₂—N(R)(R), —N(R)(R), —C(O)—N(R)(R), —N(R)—C(O)R,—N(R)—C(O)OR, —(C₁-C₆ alkylene)-N(R)—C(O)R, —N(R)S(O)₂R, and—P(O)(R)(R), wherein each of alkyl, alkenyl, alkynyl, alkoxy,heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl,heterocyclyl, and heterocyclylalkyl is independently substituted with0-5 occurrences of R^(a), or two R¹ or two R⁷ are taken together withthe carbon atoms to which they are attached form a cycloalkyl orheterocyclyl ring independently substituted with 0-5 occurrences ofR^(b); each of R², R^(3a), R^(3b), R⁴, R^(8a), and R^(8b) isindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo,hydroxyl, C₁-C₆ heteroalkyl, and —N(R)(R), wherein each alkyl, alkoxy,and heteroalkyl is optionally and independently substituted with 0-5occurrences of R^(a); each of R⁵ and R⁹ is independently hydrogen, C₁-C₆alkyl, or C₁-C₆ heteroalkyl, wherein each alkyl and heteroalkyl isoptionally and independently substituted with 0-5 occurrences of R^(a);R⁶ is selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, C₁-C₆heteroalkyl, and —N(R)(R), wherein each alkyl, alkoxy, and heteroalkylis optionally and independently substituted with 0-5 occurrences ofR^(a); each R is independently selected from hydrogen, hydroxyl, halo,thiol, C₁-C₆ alkyl, C₁-C₆ thioalkyl, C₁-C₆ alkoxy, C₁-C₆ heteroalkyl,cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, andheterocyclylalkyl, wherein each of alkyl, thioalkyl, alkoxy,heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl,and heterocyclylalkyl is independently substituted with 0-5 occurrencesof R^(a), or two R together with the atom(s) to which they are attachedform a cycloalkyl or heterocyclyl ring independently substituted with0-5 occurrences of R^(b); each R^(a) and each R^(b) is independentlyselected from C₁-C₆ alkyl, halo, hydroxyl, C₁-C₆ heteroalkyl, C₁-C₆alkoxy, cycloalkyl, heterocyclyl, and cyano, wherein each of alkyl,heteroalkyl, alkoxy, cycloalkyl, and heterocyclyl is independentlysubstituted with 0-5 occurrences of R′; each R′ is independentlyselected from C₁-C₆ alkyl, C₁-C₆ heteroalkyl, halo, hydroxyl,cycloalkyl, and cyano, or two R′ together with the atom(s) to which theyare attached form a cycloalkyl or heterocyclyl ring; m is 0, 1, or 2;and n is 0, 1, 2, or
 3. 2. The compound of claim 1 or a pharmaceuticallyacceptable salt thereof, wherein m is 1; R¹ is located at the5-position; and R¹ is C₁-C₄ alkyl substituted with 0-3 occurrences ofR^(a).
 3. The compound of claim 2 or a pharmaceutically acceptable saltthereof, wherein R¹ is CH₃.
 4. The compound of any of claims 1-3 or apharmaceutically acceptable salt thereof, wherein R² is hydrogen,hydroxyl, halo, or O—C₁-C₄ alkyl.
 5. The compound of claim 4 or apharmaceutically acceptable salt thereof, wherein R² is selected fromhydrogen, hydroxyl, fluoro, and OCH₃.
 6. The compound of claim 1 or apharmaceutically acceptable salt thereof, wherein each of R^(3a),R^(3b), R^(8a), and R^(8b) is hydrogen.
 7. The compound of claim 1 or apharmaceutically acceptable salt thereof, wherein R⁴ is selected fromhydrogen, C₁-C₄ alkyl, and O—C₁-C₄ alkyl, wherein each alkyl portion ofR⁴ is substituted with 0-3 occurrences of R^(a).
 8. The compound ofclaim 7 or a pharmaceutically acceptable salt thereof, wherein R⁴ ishydrogen, CH₃, or OCH₃.
 9. The compound of claim 1 or a pharmaceuticallyacceptable salt thereof, wherein R⁵ is hydrogen or C₁-C₄ alkylsubstituted with 0-3 occurrences of R⁸.
 10. The compound of claim 9 or apharmaceutically acceptable salt thereof, wherein R⁵ is hydrogen or CH₃.11. The compound of claim 1 or a pharmaceutically acceptable saltthereof, wherein each R⁶ is independently hydrogen, halo, or C₁-C₄ alkylsubstituted with 0-3 occurrences of R^(a).
 12. The compound of claim 11or a pharmaceutically acceptable salt thereof, wherein each R⁶ isindependently hydrogen, chloro, or CH₃.
 13. The compound of claim 1 or apharmaceutically acceptable salt thereof, wherein ring A is a 6-memberedmonocyclic heteroaryl comprising at least one nitrogen ring atom. 14.The compound of claim 13 or a pharmaceutically acceptable salt thereof,wherein ring A is

or


15. The compound of claim 1 or a pharmaceutically acceptable saltthereof, wherein n is 1; and R⁷ is pyrazol-1-yl substituted with 0-3occurrences of R^(a).
 16. The compound of claim 15 or a pharmaceuticallyacceptable salt thereof, wherein R⁷ is 4-fluoropyrazol-1-yl orpyrazol-1-yl.
 17. The compound of claim 1 or a pharmaceuticallyacceptable salt thereof, wherein R⁹ is hydrogen or C₁-C₄ alkylsubstituted with 0-3 occurrences of R^(a).
 18. The compound of claim 17or a pharmaceutically acceptable salt thereof, wherein R⁹ is hydrogen ormethyl.
 19. The compound of claim 1 having the structural Formula (Ia):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X²,R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m, and n areas defined as for structural Formula (I).
 20. The compound of claim 1having the structural Formula (Ib):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X²,R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m, and n areas defined as for structural Formula (I).
 21. A compound having thestructural Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: X¹ is N, CH, orC(halo); X² is N or CH; X³ is N or CH; R¹² is selected from hydrogen,hydroxyl, halo, and optionally substituted O—C₁-C₄ alkyl; R¹⁴ ishydrogen, optionally substituted C₁-C₄ alkyl, or optionally substitutedO—C₁-C₄ alkyl; R¹⁵ is hydrogen or optionally substituted C₁-C₄ alkyl;R¹⁶ is hydrogen or optionally substituted C₁-C₄ alkyl; R¹⁷ is hydrogenor halo; and R¹⁹ is hydrogen or optionally substituted C₁-C₄ alkyl. 22.The compound of claim 21 or a pharmaceutically acceptable salt thereof,wherein: X¹ is N, CH, or C(Cl); R¹² is selected from hydrogen, hydroxyl,fluoro, and O—CH₃; R¹⁴ is hydrogen, CH₃, or OCH₃; R¹⁵ is hydrogen orCH₃; R¹⁶ is hydrogen or CH₃; R¹⁷ is hydrogen or fluoro; and R¹⁹ ishydrogen or CH₃.
 23. The compound of claim 22 or a pharmaceuticallyacceptable salt thereof, wherein when X¹ is CH, R¹⁶ is CH₃.
 24. Thecompound of claim 21 having the structural Formula (IIa):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹²,R¹⁴, R¹⁵, R¹⁶, R^(17a), R^(17b), R^(17c), R^(18a), R^(18b), and R¹⁹ areas defined as for structural Formula (II).
 25. The compound of claim 3having the structural Formula (IIb):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹²,R^(13a), R^(13b), R¹⁴, R¹⁵, R¹⁶, R^(17a), R^(17b), R^(17c), R^(18a),R^(18b), and R¹⁹ are as defined as for structural Formula (II).
 26. Acompound chosen from:

and pharmaceutically acceptable salts thereof.
 27. A method for treatinga subject suffering from non-small cell lung cancer, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of claim 1 or a pharmaceutically acceptable saltthereof.
 28. A method for treating a subject suffering from papillarythyroid cancer, the method comprising administering to the subject atherapeutically effective amount of a compound of claim 1 or apharmaceutically acceptable salt thereof.
 29. A method for treating asubject suffering from medullary thyroid cancer, the method comprisingadministering to the subject a therapeutically effective amount of acompound of claim 1 or a pharmaceutically acceptable salt thereof.
 30. Amethod for treating a subject suffering from colorectal cancer, themethod comprising administering to the subject a therapeuticallyeffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 31. A method for treating a subject sufferingfrom multiple endocrine neoplasia, the method comprising administeringto the subject a therapeutically effective amount of a compound of claim1 or a pharmaceutically acceptable salt thereof.
 32. A pharmaceuticalcomposition comprising: a pharmaceutically acceptable carrier; and atleast one compound chosen from compounds of claim 1 or 26 andpharmaceutically acceptable salts thereof.
 33. The compound

or a pharmaceutically acceptable salt thereof.
 34. The compound

or a pharmaceutically acceptable salt thereof.
 35. The compound

or a pharmaceutically acceptable salt thereof.
 36. The compound

or a pharmaceutically acceptable salt thereof.
 37. The compound

or a pharmaceutically acceptable salt thereof.
 38. The compound

or a pharmaceutically acceptable salt thereof.
 39. The compound

or a pharmaceutically acceptable salt thereof.
 40. The compound

or a pharmaceutically acceptable salt thereof.
 41. The compound

or a pharmaceutically acceptable salt thereof.
 42. The compound

or a pharmaceutically acceptable salt thereof.
 43. The compound

or a pharmaceutically acceptable salt thereof.
 44. The compound

or a pharmaceutically acceptable salt thereof.
 45. The compound

or a pharmaceutically acceptable salt thereof.
 46. The compound

or a pharmaceutically acceptable salt thereof.
 47. The compound

or a pharmaceutically acceptable salt thereof.